CA2229749A1

CA2229749A1 - Chimeric antiviral agents which incorporate rev binding nucleic acids

Info

Publication number: CA2229749A1
Application number: CA002229749A
Authority: CA
Inventors: Flossie Wong-Staal; Mang Yu; Osamu Yamada; Gunter Kraus
Original assignee: Individual
Current assignee: University of California
Priority date: 1995-08-25
Filing date: 1996-08-09
Publication date: 1997-03-06
Also published as: EP0865282A4; US5958768A; WO1997007808A1; EP0865282A1; AU702266B2; NZ315890A; AU6770696A

Abstract

Methods and compositions for the treatment and diagnosis of viral infections are provided. These methods and compositions utilize the ability of Rev binding nucleic acids such as the SL II sequence from the HIV-1 Rev Response Element (RRE) to target therapeutic agents to the same sub-cellular location as viruses which contain RRE sequences. The use of the compositions of the invention as components of diagnostic assays, as prophylactic reagents, and in gene therapy vectors is also described.

Description

W O 97/07808 PCT~US96/12991 CHIMERIC ANTIVIRAL AGENTS WHICH INCORPORATE
REV BINDING NUCLEIC ACIDS

This invention was made with Government support under Grant No. AI36612 awarded by the National ~ 5 Institutes of Health. The Government has certain rights in this invention.
R~r~oUND OF THE lNv~llON
The primate lentiviruses, including human immunodeficiency virus (HIV) type 1 (HIV-1), and type 2 (HIV-2) and SIV are genetically, structurally and functionally similar. HIV-1 and HIV-2 are genetically related, antigenically cross reactive, and share a common cellular receptor (CD4). See, Rosenburg and Fauci (1993) in Fundamental Im-munology~ Third Edition Paul (ed) Raven Press, Ltd., New York (Rosenburg and Fauci 1) and the re~erences therein for an overview of HIV infection. Due to the pandemic spread of HIV-1 (and increasingly, HIV-2), an intense world-wide effort to unravel the molecular mechanisms and life cycle of these viruses is underway.
It is now clear that the life cycle of these viruses provide many potential targets for inhibition by gene therapy, including cellular expression of transdominant mutant gag and env nucleic acids to interfere with virus entry, TAR (the binding site for tat, which is typically required for transactivation) decoys to inhibit transcription and trans activation, and RRE (the binding site Rev; i.e., Rev Response Element) decoys and transdominant Rev mutants to inhibit RNA processing.
See, Wong-Staal et al., PCT/US94/05700; Rosenburg and Fauci (1993) in Fundamental Tmm~nology, Third Edition Paul (ed) Raven Press, Ltd., New York and the references therein for an overview of HIV infection and the HIV life cycle, gene therapy vectors utilizing ribozymes, antisense molecules, decoy genes, transdominant genes and suicide genes, including retroviruses. See also, Yu et al., Gene Therapy (1994) 1:13-26 Antisense and ribozyme W O 97/07808 PCT~US96/12991 therapeutic agents are o~ increasing importance in the treatment and prevention o~ HIV in~ection.
Antisense gene therapeutic agents and ribozymes are entering clinical trials as gene therapeutic agents ~or the treatment o~ HIV in~ection. Ribozymes are particularly potent therapeutic agents because (i) as RNA
molecules, they are not likely to induce host immunity that eliminates the transduced cells; (ii) although they resemble antisense molecules in their sequence speci~ic recognition of target RNA, their ability to cleave the target RNA catalytically renders them more e~icient than simple anti-sense molecules; and (iii) they can potentially cleave both a~erent and e~erent viral RNA, and therefore inhibit ~oth preintegration and postintegration steps of the virus replication cycle. T-cell lines (Yamada et al., Gene Therapy (1994) 1:38-45) and primary lymphocytes (Leavitt et al., Hum. Ge~e Ther.
(1994) 5:1115-1120) transduced with retroviral vectors expressing anti-HIV hairpin ribozymes are resistant to exogenous in~ection with diverse strains o~ HIV-1.
Furthermore, macrophages derived from primary CD34 hematopoietic stem/progenitor cells were also resistant to challenge with a macrophage tropic strain of HIV-1 (Yu e~ al., Virology (1995) 206:381-386).
Because of the dramatic potential of gene therapy, constructs and methods which improve the e~icacy o~ viral inhibitors used in gene therapy are o~
increasing importance. The present invention provides methods and compositions which are optionally combined with other viral inhibitors, compounds and methods to provide cells with enhanced viral resistance. The present invention also provides diagnostic reagents and methods, and kits based upon the compositions and methods o~ the invention.

W O 97t07808 PCTAJS96/12991 SU~UARY OF THE lNV ~:N'LlON
The present invention results from the discovery that the stem loop two (SL II) sequence o~ the RRE is an e~ective viral inhibitor, and that fusion molecules which comprise the SL II sequence in conjunction with an additional viral inhibitor elements are more e~ective than the inhibitor elements alone. In particular, ribozymes which comprise the SL II sequence are catalytically active, and provide greater viral protection than similar ribozymes which lack SL II
nucleic acids.
Furthermore, it is now discovered that the SL
II sequence is bi~unctional. As shown herein, the sequence is an e~ective Rev decoy, and, in addition, was shown to be su~ficient to direct cellular localization o~
bi~unctional viral inhibitors along the same cellular pathway as nucleic acids containing ~ull-length RRE
sequences (e.g., viral RNAs such as an HIV RNA).
Sequences such as SL II which bind Rev are particularly potent molecular decoys, because the Rev protein multimerizes at a Rev binding site, allowing a single Rev binding nucleic acid to act as a decoy ~or multiple copies of the Rev protein. It is also discovered that the RRE sequence acts as a molecular decoy, and can target viral inhibitors to their target viruses.
However, quite surprisingly, it is ~urther discovered that the ~ull-length RRE sequence is not stably expressed in cells over time, due to cytotoxicity, making it less suitable than RRE subsequences such as the SL II sequence as a viral inhibitor in general, and as a molecular decoy in particular.
A Fusion molecules containing Rev binding nucleic acids such as SL II nucleic acids were shown to function as Rev decoys, while preserving the activity o~ the anti-viral inhibitor to which the SL II sequence was coupled (~or example, ribozymes comprising the RRE and SL II
sequences remain catalytically active). In addition, the W O 97/07808 PCT~US96/12991 activity of the anti-viral inhibitor was enhanced due to colocalization o~ the inhibitor and the virus, i . e., with the localization of the anti-viral inhibitor being mediated through the SL II nucleic acid portion o~ the molecule. Accordingly, in one class o~ embodiments, the present invention provides a class o~ inhibitors which inhibit viruses which are bound by Rev. Such viruses typically have a Rev binding nucleic acid such as an SL
II sequence in their genome (e.g., HIV) e . g., as part of an RRE sequence, or a sequence with similar secondary structure. The inhibitors include nucleic acids with the SL II sequence (typically the nucleic acid is an RNA, or a nucleic acid which encodes an RNA), which act, inter alia, as molecular decoy molecules ~or Rev.
In addition, in many embodiments, the inhibitors o~ the present invention ~urther comprise an additional moiety or moieties with a separate anti-viral activity. This additional anti-viral activity is enhanced by the addition o~ an SL II nucleic acid, or other Rev-binding nucleic acid which causes the inhibitor to travel along the same localization pathway as the virus, providing enhanced opportunities to interact with (and there~ore inhibit) the viral nucleic acid. Although the viral inhibitor is typically a nucleic acid, other con~igurations are also desirable. Any viral inhibitor which interacts with viral nucleic acids bene~it ~rom the addition o~ the SL II nucleic acid, because the inhibitor molecule is co-localized with the viral nucleic acids.
Furthermore, multiple SL II sequences can be used in combination to enhance the decoy and targeting e~ect o~
the sequences. Thus, in one pre~erred embodiment, the inhibitor comprises a plurality o~ SL II sequences (i.e., 2 or more SL II sequences). Moreover, the SL II sequence protects RNA nucleic acids ~rom degradation by cellular nucleases by virtue o~ its secondary structure. Thus, in one pre~erred embodiment, the Rev binding sequence is attached at either the 3~ or 5~ terminal o~ an RNA (or W O 97/07808 PCT~US96/12991 nucleic acid encoding an RNA), or both, to protect the RNA ~rom degradation. For example, where a viral inhibitor comprises a ribozyme, a Rev binding nucleic acid such as the SL II se~uence is attached to either the 3~ or 5' end of the ribozyme, or both, to inhibit degradation o~ the ribozyme Anti-sense nucleic acids can be used in the inhibitors of the present invention. In one particularly preferred class of embodiments of the invention, the inhibitor includes a ribozyme. Typically, the ribozyme cleaves a viral nucleic acid, although non-functional "ribozymes~ which optionally act as anti-sense molecules are also contemplated. In one preferred embodiment, the inhibitor is a chimeric nucleic acid which includes a ribozyme such as a hairpin ribozyme, in addition to a Rev binding nucleic acid such as the SL II nucleic acid. In one preferred class of embodiments, the Rev-binding virus is an HIV virus, and the inhibitor includes a ribozyme which catalytically cleaves HIV nucleic acids.
In one class of embodiments of the invention, the invention provides viral inhibitors which comprise or encode ribozymes. In preferred embodiments of the invention, the ribozymes comprise Rev binding nucleic acids, typically at either the 5' or 3~ region of the ribozyme. The ribozyme optionally comprises multiple Rev binding nucleic acids, which are at either end of the ribozyme, or both ends of the ribozyme, or in tandem at either end of the ribozyme or at both ends of the ribozyme. Typically, the viral inhibitors comprise a recombinant expression cassette, o~ten in a gene therapy vector for transduction of cells. Typically, the gene therapy vector is designed to transduce mammalian cells with the inhibitors of the invention. Exemplar gene therapy vectors are based on retroviruses such as HIV
viruses, SIV viruses, murine retroviruses, or adeno associated viruses (AAVs).

W O 97/07808 PCTrUS96/12991 When the inhibitors of the invention are used as therapeutic agents, e . g., to provide ~e-sistance to a cell against HIV infection, the inhibitors are typically placed into a gene therapy vector such as a retroviral 5 ( e. g , HIV, SIV or MuLV) or an AAV-derived vector for transduction of a target cell upon which resistance to viral infection or replication is to be conferred. Such vectors can be used to transduce cells in vi tro, ex vivo, or in vivo. Thus, the present invention provides cells which express the inhibitors of the invention in vi tro, or in vivo. Exemplar cells include cells which express the CD4 receptor on their cell surface ( e. g., when the gene therapy vector recognizes CD4; cells, e.g., where the vector is encapsulated in an HIV capsid or envelope~, such as monocytes, lymphocytes and macrophage. The cells exist as individual cells, e g., in a cell culture, or as part of a tissue, e . g., in tissue cultures or organs, or in whole organisms, such as m~mm~ 1 S ( including primates such as humans and macaques).
The inhibitors and nucleic acids encoding the inhibitors of the invention can be incorporated into many other types of cells as well. For instance, where the inhibitor is an RNA molecule, the RNA or the corresponding nucleic acid can be cloned into a variety 25 of recombinant cells, including prokaryotes and eukaryotes. Where non-retroviral gene therapy vectors are used, the inhibitors are present in cell types transduced by the vector. For instance, AAV vectors infect most known eukaryotic cells. Thus, AAV vectors are a preferred gene therapy vector for the incorporation o~ the inhibitors of the invention into cells, particularly where the cells are present in a whole animal (e.g., a m~mm~l ) . Organ speci~ic gene therapy vectors are also known. For instance, hepatocyte virus vectors target the liver, due to the specificity of hepatocyte viruses for the liver. As already described, HIV-based vectors target CD4~ cells in vivo.

W O 97/07808 PCTnUS96/12991 The methods of the present invention provide means for inhibiting the growth, replication and expression of Rev-binding viruses in cells. These methods operate by transducing cells with an inhibitor of the invention. In one preferred class of embodiments, the methods of the invention inhibit a Rev-binding virus in a m~mm~l . In a most preferred embodiment, the methods of the invention are used to inhibit an HIV virus in a human.
Most commonly in the methods of the invention, the inhibitor is introduced into a target cell using a gene therapy vector, such as a retroviral vector, or an AAV based vector. The cells are optionally in vitro cells, such as cultured cells from a blood bank, ex vivo cells, such as CD4~ cells isolated from a mammal, or in vivo cells, i. e., where the gene therapy vector transduces cells in a whole organism, such as a m~mm~ 1 (including a human). Such gene therapy vectors include vectors with the HIV packaging site (e.g., HIV-l ~) and the AAV packaging site in the AAV inverted terminal repeat (ITR).
In one embodiment, the present invention provides methods for the detection of Rev-binding viral infections. In these methods, cells are monitored for the presence of Rev in an in vitro binding assay, using an inhibitor of the invention which includes an SL II
nucleic acid. For instance, a gel mobility-shift assay using a radio-labeled SL II nucleic acid can be used to detect Rev in a cell extract, providing an indication that the cells used to make the extract are infected with a virus ( e. g., HIV) which encodes Rev. In another diagnostic embodiment, cells suspected of being infected with a Rev-binding virus are transduced with an inhibitor of the invention. Increased survival compared to an untransduced control is diagnostic for the presence of a Rev-binding protein.

The compositions o~ the invention ~urther provide a prophylactic utility. The safety o~ handling and maintaining cell cultures is enhanced by incorporating the inhibitors o~ the invention into the cells o~ the cell culture, because the cells are rendered resistant to pathogenic viruses such as HIV. Because the cells are less likely to be in~ected with a pathogenic virus, workers handling the cells are less likely to contract the virus ~rom the cell culture.
The compositions and methods o~ the invention can be incorporated into kits ~or the treatment and diagnosis o~ Rev-binding viral infections. Typically, these kits include a container and a nucleic acid or the invention. The kits optionally include additional components such as instructional materials, reagents ~or cellular trans~ection and control cells.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. panel (A) is a schematic representation o~ the retroviral vectors expressing anti-HIV-1 ribozyme (Rz) and stem-loop II sequences (SL II) of the HIV-l RRE. The ~usion RNA is driven by an internal human tRNAVal promoter. Figure 1, panels (B) and (C) show the inhibition o~ p24 antigen expression a~ter challenge with HIV-1 SF2 at a M.O.I. o~ 0.01. (panel B) MOY-1 cells (expressing anti-Rev ribozyme), MSLOY-1 cells (expressing anti-Rev ribozyme linked to SL II), and parental Molt-4 /8 cells were in~ected with SF2 (panel C) MMJT (expressing anti-U5 ribozyme), MdMJT (expressing disabled anti-U5 ribozyme), MSLMJT (expressing anti-U5 ribozyme linked to SL II), and Molt-4/8 cells were in~ected with SF2. Culture supernatants were used ~or measurement o~ HIV-1 p24 antigen. ~: Molt-4/8; ~ : MOY-1; ~ : MSLOY-1; ~: MMJT; ~: MSLMJT; ~: MdMJT.

Figure 2 Panels A, B, C and D shows ribozyme expression levels in stable cell lines. Total cellular RNA from MMJT(A), MdMJT(B), MSLMJT(C), and MSLdMJT(D) cultured for 25 weeks after initial trans~ection was subjected to RT-PCR amplification in the presence of different amounts of competitor RNA. Ten ~l of each PCR product was loaded onto a 5~ low melting agarose gel and stained with ethidium bromide. Video images of the gel were inverted with Adobe Photoshop v3Ø The number of copies of competitor RNA added to each PCR reaction was as follows:
Lanes 1: 108; 2: 10'; 3: loG; 4: 105; 5: 0.

Figure 3, panels A and B show the inhibition of p24 expression in cell-cell transmission of HIV-l. 105 MLNL6 (-), MMJT(~), MdMJT(-), MSLMJT(~),or MSLdMJT(-) cells were suspended in 1 ml of lOso FCS supplemented RPMI 1640 with (A). 100 (1000 : 1 uninfected to infected cells) or (B). 1000 cells (100 : 1) of Jurkat cells chronically infected with HXB2. Four days after infection, the cells were split to adjust the cell concentration to 2 X 105 cells/ml, and further split 1 to 5 every 3 days thereafter. The culture supernatants were used for measurement of p24 antigen level.

Figure 4, panel~ A and B show reduction of provlral DNA
burden during a first round infection. After in~ection o~ MMJT, MdMJT, MSLMJT, or MSLdMJT cells with HIV-l HXB2 for 7 hrs, cell lysates were prepared from the infected cells to provide template DNAs for quantitative competitive PCR. The PCR was carried out using a 32P-end-labeled-SK29/SK30 primer pair derived ~rom the HIV-l LTR
in the presence of different concentrations of competitor DNA. The expected sizes of the amplified products were 105 bp and 123 bp, respectively, for the test and competitor DNA. After PCR, 3 ~l each of the PCR products was loaded on 8~ polyacrylamide gel, electrophoresed for 16 hours and autoradiographed (panel A). Images of the W O 97/07808 PCT~US96/12991 gel was scanned by Twain Scan Duo 600 (Mustek) and analyzed using NIH image v.1.54 by THE Macintosh computer (panel B). Ratio C/S; ratio of the signal intensity of the products o~ the competitor DNA and sample DNA.
Figure 5, Panels A, and B represent example sequences for SL II, and the RRE sequence (SEQ ID NO.: 1 and SEQ ID
NO.:2, respectively).

Figure 6, Panels A and B represent ribozymes which comprise Rev binding nucleic acids. Panel A is a representation of a ribozyme which comprises an SL II
nucleic acid, and which cleaves the U5 region of HIV-1.
Panel B is a similar ribozyme with the SL II sequence at the 3' terminus instead of the 5' terminus as shown in panel A. Lower case nucleic acids represent restriction sites. Panels A and B represent SEQ ID NO.: 3 and SEQ ID
NO.: 4, respectively.

Figure 7, Panels A, B and C represent ribozymes which comprise Rev binding nucleic acids. Panel A represents a ribozyme which cleaves HIV-1 nucleic acids in the env/rev region, with a 5~ Rev binding nucleic acid corresponding to the HIV-1 RRE. Panel s represents a similar env/rev ribozyme, with 5' SL II nucleic acid.
Panel C represents a similar env/rev ribozyme with 5' and 3' S~ II nucleic acids. Panels A, B, and C represent SEQ
ID NO.:5, SEQ ID NO.: 6 and SEQ ID NO.: 7, respectively.

D~:r~ lONS
Unless de~ined otherwise, all technical and scienti~ic terms used herein have the same m~n~ng as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al . ( 1994 ) Dictionary of Micro~iology and Molecular Biology, second edition, John Wiley and Sons (New York) provides one of skill with a general dictionary of many of the terms used CA 02229749 l998-02-l7 W O 97/07808 PCTrUS96/12991 in this invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are de~ined below.
A "Rev-binding virus" is a virus or derivative thereof, which comprises nucleic acid sequence elements necessary and sufficient to direct binding of a Rev protein. Most typically, the virus will be a primate lentivirus such as an HIV virus which comprises a Rev response element (RRE), or a subsequence thereof, such as the SL II sequence from HIV-1. Many allelic and strain variants of the Rev protein and the RRE are known for primate lentiviruses, particularly HIV viruses. In addition, the SL II sequence, optionally in conjunction with other RRE sequences can easily be engineered into any virus, viral vector or nucleic acid encoded by either the virus or vector using standard recombinant techniques described herein. In this way, any virus or vector can be converted into a Rev-binding virus.
A Rev binding se~uence is a nucleic acid which specifically binds to Rev in vitro or in vivo (typically an RNA), or to a nucleic acid which encodes a nucleic acid which binds to Rev in vitro or in vivo (i.e., an RNA
or a DNA). An Example of a Rev binding nucleic acid is the RNA corresponding to an SL II nucleic acid, described herein. The RRE also binds to Rev. Several papers describe in vitro binding assays for monitoring Rev binding, including Wong-Staal et al. (1991) Viral And Cellular Factors that Bind to the Rev Response Element in Genetic Structure and Reaulation of HIV (Haseltine and Wong-Staal eds.; part of the Harvard AIDS Institute Series on Gene Regulation of Human Retroviruses, Volume 1), pages 311-322 and the references cited therein, which describe gel mobility-shift assays and footprinting CA 02229749 l99X-02-17 assays ~or the detection o~ Rev in biological samples, including human blood.
An SL II nucleic acid is a nucleic acid which comprises the stem loop two region (SL II) o~ the HIV
RRE, or a conservatively modi~ied variation thereo~. SEQ
ID NO 1 provides an example SL II sequence.
An "inhibitor" or "viral inhibitor~ is most typically a nucleic acid which encodes an active anti-viral agent, or is itsel~ an anti-viral agent. Thus, in one class o~ embodiments, the inhibitor is a "direct inhibitor," i.e., the inhibitor acts directly on a viral component to inhibit the in~ection, replication, integration or growth o~ the virus in the cell. For instance, in one particularly pre~erred embodiment, the inhibitor comprises a trans-active ribozyme which cleaves a Rev-binding virus nucleic acid ( e . g., an HIV
transcript). In this con~iguration, the inhibitor is typically an RNA molecule with catalytic nuclease activity. In another class o~ embodiments, the inhibitor is an ~indirect inhibitor," i e., the inhibitor encodes the direct inhibitor. For instance, in one pre~erred embodiment, the inhibitor is part o~ a gene therapy vector, which, when expressed, produces an anti-viral RNA
which includes an SL II molecular decoy. For example, in one pre~erred embodiment, the inhibitor is a transcription cassette which is encoded by a gene therapy vector which is used to trans~ect a cell, where the transcription cassette expresses a nucleic acid which encodes an SL II nucleic acid, and, optionally, a ribozyme or antisense molecule which inhibits the ability o~ a rev-binding virus such as HIV-1 to replicate in the cell. An inhibitor '~encodes" a direct inhibitor such as an active ribozyme, RNA molecular decoy, or anti-sense RNA i~ it contains either the sense or anti-sense coding or complementary nucleic acid which corresponds to the direct inhibitor. By convention, direct inhibitor RNAs such as ribozymes are typically listed as their W O 97/07808 PCT~US96/12991 corresponding DNA sequences. For instance, in SEQ ID
NOs. 1-7 herein, sequences are listed 5' to 3' as the DNA
which corresponds directly to the encoded RNA. This is done to simplify visualization of the corresponding active RNA, which is equivalent to the given sequence with the T residues replaced b~U re~idues.
Although the inhibitor is typically an RNA, or a nucleic acid which encodes the RNA (i.e., DNA or RNA), other configurations are also possible. For instance, in one embodiment, the inhibitor includes protein or other elements with anti-viral activity. For example, in one embodiment, the inhibitor comprises an SL II nucleic acid and a bound Rev protein (e.g., an endogenous protein from the cell, or a Rev protein from an invading virus). In one embodiment, the inhibitor optionally includes nucleic acids which encode separate protein binding sites such as the TAR site for Tat binding, and the bound protein.
"Viral inhibition" refers to the ability of a construct to inhibit the infection, growth, integration, or replication of a virus in a cell. Inhibition is typically measured by monitoring changes in a cell's viral load (i.e., the number of viruses and/or viral proteins or nucleic acids present in the cell, cell culture, or organism) or by monitoring resistance by a call, cell culture, or organism to infection.
A "targeted anti-HIV chimeric nucleic acid"
refers either to a nucleic acid which encodes an SL II
nucleic acid and an anti- Rev-binding virus agent (such as a ribozyme or an anti-sense molecule which inhibits the Rev-binding virus), or to the encoded nucleic acid.
Thus, in one embodiment, the targeted anti-HIV chimeric nucleic acid is part of a gene therapy vector which encodes an SL II nucleic acid and a Rev-binding viral element. In a second embodiment, the targeted anti-HIV
chimeric nucleic acid is a nucleic acid (typically an RNA) which includes an SL II sequence and an anti-Rev-WO 97/07808 PCT~US96/12991 binding viral agent (e.g., ribozyme or anti-sense) sequence.
Ribozymes are typically either "cis-ribozymes~ -or trans-ribozymes. Cis ribozymes cleave the nucleic acid which they are part of, whereas trans-ribozymes catalytically cleave nucleic acids which they are not covalently linked to. A ribozyme optionally has both cis- and trans- activity, and in some embodiments, a cis-ribozyme is converted into a trans ribozyme after a cis cleavage event.
The term ~identical" in the context of two nucleic acids refers to the nucleotide residues in the two sequences which are the same when aligned ~or maximum correspondence.
Methods o~ alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981~ Adv.
Appl. Math. 2: 482; by the homology alignment algorithm o~ Needleman and Wunsch (1970) J. Mol. Biol. 48: 443; by the search ~or similarity method o~ Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444; by computerized implementations of these algorithms (including, but not limited to CLUSTAL in ~he PC/Gene program by Intelligenetics, Mountain View, California, GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin, USA); the CLUSTAL
program is well described by Higgins and Sharp (1988) Gene, 73: 237-244 and Higgins and Sharp (1989) CABIOS 5:
151-153; Corpet, et al. (1988) Nucleic Acids Research 16, 10881-90; Huang, et al. (1992) Computer Applications in the Biosciences 8, 155-65, and Pearson, et al. (1994) Methods in Molecular Biology 24, 307-31. Alignment is also often performed by inspection and manual adjustment of the sequences.

W O 97/07808 PCT~US96/12991 The terms "isolated~' or "biologically pure"
refer to material which is substantially or essentially -free from components which normally accompany it as found in its native state. The isolated nucleic acids of this -5 invention do not contain materials normally associated with their in si tu environment, in particular, nuclear, cytosolic or membrane associated proteins or nucleic acids other than those nucleic acids which are indicated.
The term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly provides the complementary sequence thereof, as well as the sequence explicitly indicated.
The term "operably linked" refers to functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription ~actor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
The term "recombinant"Swhen used with reference to a cell indicates that the cell replicates or expresses a nucleic acid, or expresses a peptide or protein encoded by a nucleic acid whose origin is exogenous to the cell.
Recombinant cells can express genes that are not found within the native (non-recombinant) ~orm of the cell.
Recombinant cells can also express genes found in the native form o~ the cell wherein the genes are re-introduced into the cell by artificial means.
A "recombinant expression cassette" or simply an "expression cassette" is a nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements which permit transcription o~ a particular W O 97/07808 PCT~US96/12991 nucleic acid in a cell. The recombinant expression cassette can be part of a plasmid, virus, or nucleic acid fragment. Typically, the recombinant expression cassette includes a nucleic acid to be transcribed, and a promoter. In some embodiments, the expression cassette also includes, e.g., an origin of replication, and/or chromosome integration elements (e.g., an AAV ITR, or retroviral LTR).
The term "subsequence" in the context of a lo particular nucleic acid se~uence re~ers to a region of the nucleic acid equal to or smaller than the specified nucleic acid.

DET~TT-~n DISCUSSION OF THE l~v~-LlON AND DESCRIPTION OF

~ ~RED EMBODIMENTS
The molecular receptor for HIV is the surface glycoprotein CD4 found mainly on a subset of T cells, monocytes, macrophage and some brain cells. HIV has a lipid envelope with viral antigens that bind the CD4 receptor, causing fusion of the viral membrane and the target cell membrane and release o~ the HIV capsid into the cytosol. HIV causes cell death of these immune cells, thereby disabling the immune system and eventually causiny death of the patient due to complications 2S associated with a disabled immune system. HIV infection also spreads directly from cell to cell, without an intermediate viral stage. During cell-cell transfer of HIV, a large amount of viral glycoprotein is expressed on the surface of an infected cell, which Dinds CD4 receptors on uninfected cells, causing cellular fusion.
This typically produces an abnormal multinucleate syncytial cell in which HIV is replicated and normal cell functions are suppressed Recent studies of the dynamics of HIV
replication ln patients under antiviral therapy have reaffirmed the central role of the virus in disease progression, and provided a strong rationale for the WO 97/07808 PCTrUS96/12991 development of e~fective, long term antiviral therapy (Coffin, J. M. Science (1995) 267:483-489; Ho et al., Nature (1995) 373:123-6; Wei et al., Nature (1995) 373:117-22). One interesting parameter from these studies is the extremely short life span of an HIV-1 infected CD4~ lymphocyte (half life = 1-2 days), contrasting data ~rom other studies which gave an estimated lifespan of months to years for uninfected lymphocytes (Bordignon et al., Hum Gene Ther. (1993) 4:513-20). These observations are relevant for antiviral gene therapy, because an "intracellularly immunized" cell resistant to viral infection, or which suppresses viral replication will be strongly selected for in vivo.
In previous studies, the efficacy o~ several anti-HIV-1 hairpin ribozymes in inhibiting virus replication in human T cell lines was demonstrated. See, Wong-Staal et al., PCT/US94/05700; Yamada et al., Virology ( 1994) 205:121-126; Yamada et al., Gene Therapy (1994) 1:38-45; Yu et al., Proc Natl. Acad. Sci. USA
(1993) 90:6340-6344; Yu et al., Virology (1995) 206:381-386 and Yu et al. (1993) PNAS 90: 6340-6344. With an anti-U5 ribozyme which targets a highly conserved region of the HIV-1 genome, it was shown that intracellular immllnization of primary lymphocytes or hematopoietic progenitor cells could lead to resistance to both lymphotropic and macrophage tropic HIV-1 strains (Leavitt et al., Hum. Gene Ther. (1994) 5:1115-1120; Yu et al., Proc. Natl. Acad. Sci. USA (1995) 92:699-703).
To increase antiviral potency of anti-viral inhibitors such as ribozyme vectors, as well as to reduce the chance of viral resistance, we explored the possibility of adding other antiviral agents to the ribozyme constructs.
Rev, an early gene product of HIV, controls expression of the HIV-1 structural genes through binding to a Rev Response Element (RRE) present in unspliced or partially spliced viral transcripts. Rev facilitates the CA 02229749 l998-02-l7 nuclear export and utilization o~ such transcripts in the cytoplasm (Feinberg et al., Cell (1986) 46:807-817; Malim et al., Nature (1989) 338:254-257). We hypothesized that linking the RRE sequence to a ribozyme would improve e~f~icacy o~ the ribozyrne because such a molecule would be bi~unctional ( e . g., by providing a nuclease + Rev decoy e~ect). In addition, we hypothesized that ribozyme activity would also be ~acilitated by linking the ribozyme to the RRE, because the RRE would stabilize the ribozyme molecule by inhibiting degradation of the ribozyme. Furthermore, as described herein, we ~ound that binding o~ Rev to the RRE-ribozyme ~usion molecule tra~ics the ~usion molecule along the same nuclear-cytoplasmic pathway as HIV mRNA (which is Rev dependent), 15 thereby increasing the opportunity for interaction between the ribozyme and the ~IV substrate. Finally, we ~ound that binding o~ Rev to the ribozyme-substrate complex increases the turnover of the ribozyme, resulting in increased catalytic activity.
The HIV-1 RRE is 234 nt in length, predicted to form a central stem and ~ive stem-loop structures (Feinberg et al., Cell (1986) 46:807-817). The entire ~ull-length RRE sequence was inserted into a ribozyme expression cassette. For the ~irst time, such a 25 combination o~ the RRE and ribozyme was shown to produce a ~unctional ribozyme, and the construct displayed strong viral inhibition. Surprisingly, however, although cell lines expressing such ~usion RNAs demonstrated strong virus inhibition, expression o~ the ~usion RNA (RRE +
30 ribozyme) was turned oi~:E at week 15 a~ter transEection.
While not beiny bound to a particular theory, it is hypothesized that interaction between the RRE and one or more mammalian cellular proteins (which is known to occur, ~ee, Vaishnav et al., New Biol. (1991) 3:142-150) 35 induces cellular toxicity and provides negative selection for cells expressing the ~usion RNA.

W O 97/07808 PCT~US96/12991 To overcome the unexpected property o~
cytotoxicity by RRE, a minimal sequence comprising the second stem loop o~ RRE (SL II) was cloned and tested in the ribozyme constructs. An SL II-anti-U5 ribozyme ~usion RNA was shown to be persistently expressed in stable cell lines for over 25 weeks. It was also ~ound to be more e~ective in virus inhibition than the ribozyme alone, or the SL II nucleic acid linked with a disabled ribozyme ( See also, Fig. 3).
The decoy e~ect of the ~usion RNA was demonstrated by HIV-l SF2 in~ection o~ a stable cell line, MSLOY-1, expressing the SL II sequence linked to an anti-Rev ribozyme. HIV-1 SF2 is re~ractory to inhibition by the anti-Rev ribozyme because o~ a substitution at the G residue at the site o~ cleavage (Fig. lB) ( see also, Yamada et al., Virology (1994) 205:121-126). There~ore, the observed inhibitory e~ect o~ the ~usion RNA is due to the SL II sequence acting as a decoy.
Also demonstrated herein is the ribozyme activity of the ~usion RNA by showing a reduction o~
proviral DNA burden in a ~irst round in~ection ( see, e. g., Figure 4). Additionally, the ~usion RNA exerted a two ~old greater reduction in viral DNA synthesis than the ribozyme alone. Bertrand, e t al ., Emho J . ( 19 94 ) 13:2904-12 reported that adding the nucleocapsid protein o~ HIV-1, or the heterogeneous ribonucleoprotein A1 to the cleavage reaction o~ hammerhead ribozymes was able to increase binding, speci~icity, and turnover o~ the ribozymes in vitro without inhibiting cleavage, depending on the length o~ the ribozyme-substrate duplexed region.
The time-regulated nuclear export o~ Rev is correlated with protein expression ~rom RRE-containing mRNAs, and distribution o~ Rev re~lects its interaction with RRE-containing RNA and migration o~ the bound transcript ~rom the nucleolus across a solid phase o~ nucleus and nuclear membrane to the cytoplasm through a speci~ic export pathway (Luznik et al., AIDS Res. ~um. Retrovirus (1995) CA 02229749 l998-02-l7 W 097/07808 PCT~US96/12991 11:795-804). SL II i~usion RNA (like other Rev-binding nucleic acid ~usion RNAs) tra~ics through the same cellular compartments as HIV mRNA caused by the binding o~ Rev, thereby increasing the e~iciency of the ribozyme by increasing the opportunity f~or interaction between the ribozyme and the viral nucleic acid.

Viral Inhibitors Viral inhibitors o~ the invention take several ~orms. Typically, the viral inhibi~or is a nucleic acid which has direct anti-viral activity, such as a molecular decoy, anti-sense RNA or ribozyme, or indirect anti-viral activity, i.e., where the inhibitor encodes a direct anti-viral activity ( e. g., where the inhibitor encodes a ribozyme RNA, Eor example in conjunction with an SL II
sequence). The inhibitors o~ the invention typically include an SL II nucleic acid, either in its active (i . e., RNA) molecular decoy form, or in its encoded ~orm ( i . e ., in an RNA or DNA vector which encodes the active form). Thus, techniques applicable to the construction and maintenance o~ nucleic acids apply to the nucleic acid inhibitors o~ the present invention.
In pre~erred embodiments, the inhibitors o~ the invention include ribozymes, such as hairpin ribozymes 25 (see, Wong-Staal et al. WO 94/26877 and PCT/US94/05700 and the re~erences therein; see also, Yu et al . ( 1993) PNAS 90: 6340-6344; and Yu et al. (1995) Virology 206:
381-386), hammerhead ribozymes ( see, Dropulic e t al .
(1992) Journal of Virology, 66(3):1432-1441), and RNAse 30 P (see, Castanotto et al. (1994) Advances in Pharmacology Academic Press 25: 289-317). These ribozymes are constructed to target a portion o~ the Rev-binding virus' genome or nucleic acid encoded by the genome. Pre~erred target sites in HIV-1 include the U~ region, and the 35 polymerase gene.
=

CA 02229749 l998-02-l7 W097/07808 PCTrUS96/12991 Antiviral Agents: antisense nucleic acids, ribozymes, decoy nucleic acids and trans-d~min~nt - proteins Viral inhibitors optionally comprise antiviral agents. Anti-viral agents are known in the art. The literature describes such genes and their use. See, for example, Yu et al., Gene Therapy, 1:13 (1994);
Herskowitz, Nature, 329:212 (1987) and Baltimore, Nature, 335:395 (1988). Anti-viral agents which are optionally incorporated into the viral inhibitors o~ the invention include anti-sense genes, ribozymes, decoy genes, and transdominant proteins.
An antisense nucleic acid is a nucleic acid that, upon expression, hybridizes to a particular RNA
molecule, to a transcriptional promoter or to the sense strand o~ a gene. By hybridizing, the antisense nucleic acid interferes with the transcription of a complementary nucleic acid, the translation o~ an mRNA, or the function of a catalytic RNA. Antisense molecules useful in this invention include those that hybridize to viral gene transcripts. Two target sequences ~or antisense molecules are the first and second exons of the HIV genes tat and rev. Chatterjee and Wong, supra, and Marcus-Sekura ( Analytical Biochemistry (19 88) 172, 289-285) describe the use o~ anti-sense genes which block or modify gene expression.

A ribozyme is a catalytic RNA molecule that cleaves other RNA molecules having particular nucleic acid sequences. General methods for the construction of ribozymes, including hairpin ribozymes, hammerhead ribozymes, RNAse P ribozymes (i.e., ribozymes derived ~rom the naturally occurring RNAse P ribozyme ~rom prokaryotes or eukaryotes) are known in the art.
Castanotto et al (1994) Advances in Pharmacology 25: 289-317 provides and overview o~ ribozymes in general, including group I ribozymes, h~mm~rhead ribozymes, hairpin ribozymes, RNAse P, and axhead ribozymes.
Ribozymes use~ul in this invention include those that cleave viral transcripts, particularly HIV gene transcripts. Ojwang et al., Proc. Nat'1. Acad. Sci., U.S.A., 89:10802-06 (1992~; Wong-Staal et al.
(PCT/US94/05700); Ojwang et al. (1993) Proc Natl Acad Sci USA 90 :6340-6344; Yamada et al. (1994) Human Gene Therapy 1:39-45; Leavitt e~ al. (1995) Proc Natl Acad Sci USA
92:699-703; Leavitt et al. (1994) H~man Gene Therapy 5:1151-1120; Yamada et al. (1994) Virology 205:121-126, and Dropulic et al. (1992) Journal of Virology 66(3):1432-1441 provide an examples of HIV-1 speci~ic hairpin and hammerhead ribozymes.
Briefly, two types of ribozymes that are particularly use~ul in this invention include the hairpin ribozyme and the hammerhead ribozyme. The hammerhead ribozyme (see, Rossie et al. (1991) Pharmac. Ther.
50:245-254; Forster and Symons (1987) Cell 48:211-220;
Haselo~f and Gerlach (1988) Nature 328:596-600; Walbot and Bruening (1988) Nature 334:196; Haselo~ and Gerlach (1988) Nature 334:585; and Dropulic et al and Castanotto et al., and the re~erences cited therein, supra) and the hairpin ribozyme (see, e.g., Hampel et al. (1990) Nucl.
Acids Res. 18:299-304; Hempel et al., (1990) European Patent Publication No. 0 360 257; U.S. Patent No.
5,254,678, issued October 19, 1993; Wong-Staal et a7., PCT/US94/05700; Ojwang et al. (1993) Proc Natl Acad Sci USA 90:6340-6344; Yamada et al. (1994) Human Gene Therapy 1:39-45; Leavitt et al. (1995) Proc Natl Acad Sci USA
92:699-703; Leavitt et al. (1994) Human Gene Therapy 5:1151-1120; and Yamada et al. (1994) Virology 205:121-126) are catalytic molecules having antisense and endoribonucleotidase activity. Intracellular expression o~ hammerhead ribozymes and a hairpin ribozymes directed against HIV RNA has been shown to con~er signi~icant resistance to HIV in~ection.

The typical sequence requirement for cleavage by a hairpin ribozyme is an RNA sequence consisting of NNNG/CN*GUC~NNNNNNN (where N*G is the cleavage site, and where N is any of G, U, C, or A). The sequence requirement at the cleavage site for the hammerhead ribozyme is an RNA sequence consisting of NUX (where N is any of G, U, C, or A and X represents C, U or A).
Accordingly, the same target within the hairpin leader sequence, GUC, is targPtable by the hammerhead ribozyme.
The additional nucleotides of the hammerhead ribozyme or hairpin ribozyme which mediate sequence specificity, are determined by the common target ~lanking nucleotides and the hammerhead and hairpin consensus sequences.
Altman (1995) Biotechnology 13: 327-329 and the references therein describe the use of RNAse P as a therapeutic agent directed against flu virus. Similar therapeutic approaches can be used against Rev binding viruses such as HIV by incorporating RNAse P into the inhibitors of the invention.
The ribozymes of this invention and DNA
encoding the ribozymes, can be chemically synthesized as described in more detail below using methods known in the art, or prepared from a DNA molecule (that upon transcription, yields an RNA molecule) operably linked to an appropriate promoter.
A decoy nucleic acid is a nucleic acid having a sequence recognized by a regulatory nucleic acid binding protein (i.e., a transcription factor, cell trafficking factor, etc. ) . Upon expression, the transcription factor binds to the decoy nucleic acid, rather than to its natural target in the genome. Useful decoy nucleic acid sequences include any sequence to which a viral transcription ~actor binds. For instance, the TAR sequence, to which the tat protein binds, and the HIV RRE sequence (in particular the SL II sequence), to which the rev proteins binds are suitable sequences to use as decoy nucleic acids.

CA 02229749 l998-02-l7 W 097/07808 PCT~US96/12991 A transdominant protein is a protein whose phenotype, when supplied by transcomplementation, will overcome the e~ect o~ the native form of the protein.
For example, tat and rev can be mutated to retain the ability to bind to TAR and RRE, respectively, but to lack the proper regulatory function of those proteins. In particular, rev can be made transdominant by eliminating the leucine-rich domain close to the C terminus whicn is essential for proper normal regulation of transcription.
Tat transdominant proteins can be generated by mutations in the RNA binding/nuclear localization domain.
Transdominant proteins can be encoded by the inhibitors o~ the invention, for instance, in an expression cassette which also includes, e.g., the SL II molecular decoy in 15 conjunction with a ribozyme.
Examples of antisense molecules, ribozymes and decoy nucleic acids and their use can be found in Weintraub, Sci. Am. , 262:40-46 (Jan. 1990);
Marcus-Sekura, Anal. Biochem. , 172:289-95 (1988); and 20 Hasselhoff~ et al., Nature, 334:585-591 (1988), incorporated herein by re~erence.

Makinq Viral Inhibitors The present invention provides a variety o~
25 viral inhibitors as described supra. Typically, these viral inhibitors are nucleic acids such as the SL II
sequence, ribozymes against HIV or anti-sense sequences, or the corresponding nucleic acids which encode such nucleic acids.
Given the general strategy for making viral inhibitor nucleic acids of the present invention, one of skill can construct a variety o~ clones containing viral inhibitors and derivative clones. Cloning methodologies to accomplish these ends, and sequencing methods to 35 verify the sequence o~ nucleic acids are well known in the art. Examples o~ appropriate cloning and sequencing techniques, and instructions sufficient to direct persons W O 97/07808 PCT~US96/12991 of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techni~ues, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel).
Product information from manufacturers of biological reagents and experimental equipment also provide information use~ul in known biological methods. Such manufacturers include the SIGMA chemical company (Saint Louis, MO), R&D systems (Minneapolis, MN), Pharmacia LKB
Biotechnology (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL
Life Technologies, Inc. (Gaithersberg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to one of skill.
The nucleic acid compositions of this invention, whether RNA, cDNA, genomic DNA, or a hybrid o~
the various combinations, are isolated from natural sources or synthesized in vitro. The nucleic acids claimed are present in transformed or transfected whole cells, in transformed or transfected cell lysates, or in a partially purified or substantially pure form.
In vitro amplification techniques suitable for amplifying provirus sequences for use as molecular probes or generating nucleic acid fragments for subsequent subcloning are known. Examples of techniques sufficient to direct persons of skill through such in vitro amplification methods, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Q~-W O 97/07808 PCTnJS96/12991 replicase ampli~ication and other RNA polymerase mediated techniques (e.g., NASBA) are ~ound in Berger, Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Patent No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, CA (1990) (Innis); Arnheim & Levinson (October 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA
87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826;
Landegren et al., (1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117, and Sookn~n~n and Malek (1995) Biotechnology 13: 563-564.
Improved methods o~ cloning in vitro ampli~ied nucleic acids are described in Wallace et al., U. S. Pat. No.
5,426,039.
Oligonucleotides ~or use as probes, e.g., in in vitro ampli~ication methods, ~or use as gene probes, or as inhibitor components are typically synthesized chemically according to the solid phase phosphoramidite triester method descri~ed by Beaucage and Caruthers (1981), Tetrahedron Letts., 22(20):1859-1862, e.g., using an automated synthesizer, as described in Needham-VanDevanter et al. (1984) Nucleic Acids Res., 12:6159-6168. Puri~ication o~ oligonucleotides, where necessary, is typically per~ormed by either native acrylamide gel electrophoresis or by anion-exchange HPLC
as described in Pearson and Regnier (1983) ~. Chrom.
30 255:137-149. The sequence o~ the synthetic oligonucleotides can be veri~ied using the chemical degradation method o~ Maxam and Gilbert (1980) in Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology 65:499-560.
One o~ skill will recognize many ways o~
generating alterations ln a given nucleic acid sequence.
Such well-known methods include site-directed W O 97/07808 PCT~US96/12991 mutagenesis, PCR ampli~ication using degenerate oligonucleotides, exposure o~ cells containing the - nucleic acid to mutagenic agents or radiation, chemical synthesis o~ a desired oligonucleotide (e. g., in conjunction with ligation and/or cloning to generate large nucleic acids) and other well-known techniques.
See, Giliman and Smith (1979) Gene 8 :81-97; Roberts et al. (1987) Nature 328:731-734 and Sambrook, Innis, Ausbel, Berger, Needham VanDevanter and Mullis (all supra).
One o~ skill can select a desired inhibitor nucleic acid o~ the invention based upon the sequences and strategies provided herein, and upon knowledge in the art regarding primate lentiviruses generally. The life-cycle, genomic organization, developmental regulation andassociated molecular biology o~ lentiviruses such as HIV
and SIV viruses have been the ~ocus o~ over a decade of intense research. The speci~ic e~ects o~ many mutations in the primate lentiviral genome are known, and the interaction of many o~ the components of the viruses at a molecular level are known.
Polypeptides o~ the invention can be synthetically prepared in a wide variety o~ well-know ways. For instance polypeptides o~ relatively short size, can be synthesized in solution or on a solid support in accordance with conventional techniques. See, e.g., Merri~ield (1963) ~. Am. Chem. Soc. 85:2149-2154.
Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, e.g., Stewart and Young (1984) Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co. More typically, polypeptides are produced by recombinant expression o~
nucleic acid encoding the polypeptide and puri~ication, using standard techniques.

CA 02229749 l998-02-l7 W 097/07808 PCT~US96/12991 Rev Bindinq Nucleic Acids: The SL II seauence and the RRE
Rev binding nucleic acids are defined functionally to consist o~ nucleic acids which bind to Rev, or which encode nucleic acids which bind to Rev. A
variety of sequences which bind Rev are known. Different Rev proteins are known for different viruses (e.g., Rev-1 ~rom HIV-1, and Rev- 2 from HIV-2, and the sequence requirements for each are similar, though not identical.
For instance, even though the HIV- 2 Rev protein cannot substitute for the HIV-1 Rev protein by transcomplementation, both HIV-1 and HIV-2 forms of the Rev protein (Rev-1 and Rev- 2) bind to the HIV-1 RRE, with roughly equivalent sequence specificity ~or the HIV-1 stem loop-2 region (See, Garrett and Cullen (1992) J.
Virol. 66(7): 4288-4294).
The SL II region from HIV-1 (comprising 66 nt from the HIV-1 RRE) is a suitable Rev-binding nucleic acid of the invention (See, SEQ ID NO 1). The HIV-1 RRE
(See, SEQ ID NO 2; 234 nt) also binds Rev, and can be used in the inhibitors o~ the invention. However, the complete RRE is not pre~erred in applications where the inhibitor is expressed in a cell to produce viral inhibition, because expression of nucleic acids containing the complete HIV-1 RRE is down-regulated in cells, due to apparent cytotoxicity. Additional Rev binding proteins are constructed which comprise the SL II
sequence, but which lack the ~ull-length RRE sequence by cloning and expressing RRE sub-sequences which contain the SL II sequence. Indeed, the complete SL II sequence is not necessary for Rev binding, and sub-sequences of SL
II are optionally used in the inhibitors of the invention. Typically, the SL II subsequences which bind Rev comprises at least 10 consecutive nucleotides from SL
II, more typically at least about 20 consecutive nucleoctides from SL II, preferably 30 consecutive nucleotides from SL II, still more preferably 40 consecutive nucleotides ~rom SL II, ideally at least W O 97/07808 PCT~US96/12991 about 40 consecutive nucleotides from SL II, and most preferably at least about 50 consecutive nucelotides from SL II. However, larger Rev binding nucleic acids which comprise the SL II sequence are typically preferred, particularly when the inhibitors o~ the invention comprise or encode ribozymes or other RNA molecules.
This is because the secondary structure of the Rev binding site (e.g., the stem loop of SL II) protect other RNA components of the inhibitor from degradation by cellular RNAse enzymes.
Accordingly, in preferred embodiments, a Rev binding nucleic acid comprises an SL II nucleic acid.
One of skill can easily add flanking sequences from the RRE (or other heterologous nucleic acid) to the SL II
sequence by synthesizing and subcloning appropriate nucleic acids. The sequences within the RRE which result in inhibition of expression of the inhibitors containing the construct can be determined by standard deletion analysis of the RRE. Brie~ly, RRE subsequences to be tested for activity as shown in the examples below are cloned into the same vectors which were shown herein to provide inhibition of Rev binding viruses. The subsequences contain the SL II sequence plus additional flanking sequence from the RRE are combined with a ribozyme as described herein, and inhibitory effect is monitored as determined herein. Sequences which stably provide an inhibitory effect for more than 15 weeks are suitable Rev binding sites. At a given point between the 66 nt of the full-length SL II and the full length 234 nt RRE, inhibition of the expression of the inhibitor (i.e., due to cytotoxicity) is observed. If deletions (i.e., SL
II sequences + RRE flanking sequences) are made, for instance, every 10 nt, then each 10 nt subsequence between the SL II sequence and the full-length RRE can easily be monitored for resulting cytotoxicity o~ the inhibitor. Particular regions within the RRE which cause cytotoxicity are omitted from preferred embodiments.

The SL II Rev binding site is typical oE Rev binding sites, in that Rev primarily recognizes the secondary structure o~ the nucleic acid (i.e., the stem- -loop structure). Modi~ications are made to the sequence to result in ~unctionally similar (i.e., Rev-binding) sequences by modi~ying the sequence such that the secondary structure o~ the nucleic acid is retained.
This is done by altering corresponding nucleotides in the structure to yield equivalent nucleic acid hybridization in the secondary structure o~ the nucleic acid. For instance, in a region o~ the secondary structure where a C binds to a G, the two nucleotides can be reversed to yield the same overall secondary structure o~ the molecule. The ability o~ the resulting sequence to bind Rev is monitored in standard gel mobility-shi~t assays as described herein. Sequences which are altered to yield similar secondary structure to the SL II sequence, and which bind to Rev, are "conservatively modi~ied"
variations o~ the SL II sequence.
The Sequence of~ an SL II nucleic acid (SEQ ID
NO 1) is: GCACTATGGG CGCAGCCTCA ATGACGCTGA CGGTACAGGC
CAGACAATTA TTGTCTGGTA TAGTGC.
The Sequence o~ an RRE nucleic acid (SEQ ID NO
2) is GGAGCTTTGT TCCTTGGGTT CTTGGGAGCA GCAGGAAGCA
CTATGGGCGC AGCCTCAATG ACGCTGACGG TACAGGCCAG ACAATTATTG
TCTGGTATAG TGCAGCAGCA GAACAATTTG CTGAGGGCTA TTGAGGCGCA
ACAGCATCTG TTGCAACTCA CAGTCTGGGG CATCAAGCAG CTCCAAGCAA
GAATCCTAGC TGTGGAAAGA TACCTA~AGG.

Ribozymes with Rev bindin~ nucleic acids The Rev binding nucleic acids described above are incorporated into pre~erred ribozymes o~ the invention. Rev binding ribozymes such as the SL II
nucleic acid are incorporated into pre~erred ribozymes at either the 3~ or 5~ end o~ the ribozyme, or both. For instance, in one pre~erred embodiment, the ribozyme comprises an SL II nucleic acid at the 3~ terminus o~ the W O 97/07808 PCTrUS96/12991 ribozyme, and an SL II nucleic acid at the 5' terminus of the nucleic acid. Examples of Rev binding ribozymes are given in SEQ ID NOs 3-7 (Figures 6 and 7). The given nucleic acids represent the DNA ~orm of the active RNA, i.e., the sequences are the same as the active RNA, except that the U residues are substituted for T
residues. DNA vectors which encode or express the ribozymes o~ the invention typically include both sense and anti-sense strands of DNA which encode the catalytically active RNA form of the ribozyme.
In one preferred embodiment, the ribozyme targets the U5 region of HIV-1, and comprises an SL II
nucleic acid at the 5' end of the ribozyme. In this embodiment, the ribozyme has the sequence GCACTATGGG CGCAGCCTCA ATGACGCTGA CGGTACAGGC CAGACAATTA
TTGTCTGGTA TAGTGCggat ccACACAACA AGAAGGCAAC CAGAGAAACA
CACGTTGTGG TATATTACCT GGTacgcgt (SEQ ID NO.: 3). In another preferred embodiment, the anti- U5 ribozyme has the SL II nucleic acid located at the 3' end of the ribozyme: ggatccACAC AACAAGAAGG CAACCAGAGA AACACACGTT
GTGGTATATT ACCTGGTacg cgtGCACTAT GGGCGCAGCC TCAATGACGC
TGACGGTACA GGCCAGACAA TTATTGTCTG GTATAGTGC (SEQ ID NO.:
4). Note that similar ribozymes have multiple SL II
ribozymes at either the 3' or 5' end o~ the ribozyme, or both.
An example ribozyme which cleaves in the env/rev region of HIV-1 which incorporates the RRE at the 5' end of the ribozyme is provided by SEQ ID NO.: 5:
GGAG~lll~l TCCTTGGGTT CTTGGGAGCA GCAGGAAGCA CTATGGGCGC
AGCCTCAATG ACGCTGACGG TACAGGCCAG ACAATTATTG TCTGGTATAG
TGCAGCAGCA GAACAATTTG CTGAGGGCTA TTGAGGCGCA ACAGCATCTG
TTGCAACTCA CAGTCTGGGG CATCAAGCAG CTCCAAGCAA GAATCCTAGC
TGTGGAAAGA TACCTAAAGG ggatcCTAGT TCCTAGAACC AAACCAGAGA
AACACACGTT GTGGTATATT ACCTGGTacg cgt.
A preferred ribozyme which cleaves HIV-1 targets in the env/rev region and includes an SL II

W O 97/07808 PCT~US96/12991 sequence at the 5' end of the ribozyme is provided by SEQ
ID NO.: 6:
GCACTATGGG CGCAGCCTCA ATGACGCTGA CGGTACAGGC CAGACAATTA
TTGTCTGGTA TAGTGCggat cCTAGTTCCT AGAACCA~AC CAGAGA~ACA
CACGTTGTGG TATATTACCT GGTacgcgt.
A preferred ribozyme which cleaves HIV-1 nucleic acids in the env/rev region, and which comprises an SL II nucleic acid at both the 3' and 5' ends o~ the ribozyme is provided by SEQ ID NO.: 7:
GCACTATGGG CG QGCCTCA ATGACGCTGA CGGTACAGGC CAGACAATTA
TTGTCTGGTA TAGTGCggat cCTAGTTCCT AGAACCA~AC CAGAGAAACA
CACGTTGTGG TATATTACCT GGTacgcgtG CACTATGGGC GCAGCCTCAA
TGACGCTGAC GGTACAGGCC AGACAATTAT TGTCTGGTAT AGTGC.
By convention, the above ribozyme sequences are provided as DNA se~uences which have the same nucleic acid as the active RNA form of the ribozyme, except that in the RNA form, the T nucleotides are replaced with U
nucleotides. Lower case letters indicate restriction endonuclease clevage sites.
Expression and Subcloninc of Nucleic Acid Viral Inhibitors Once a nucleic acid inhibitor is synthesized or cloned, one may sub clone the inhibitor or express the inhibitor in a variety of recombinantly engineered cells known to those of skill in the art. As used herein, "expression" refers to transcription of nucleic acids, with or without subsequent translation. For instance, in most embodiments of the invention, the active form o~ the inhibitor is an RNA molecule which acts as a molecular decoy ~or Rev (through binding of Rev to SL II) and as an anti-sense or ribozyme sequence which disrupts normal viral function.
Examples of cells which are suitable for the subcloning and expression of the nucleic acid inhibitors of the invention include bacteria, yeast, filamentous ~ungi, insect (especially employing baculoviral vectors), W O 97/07808 PCTrUS96/12991 and m~mm~ ian cells. It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for cloning and expression of the viral inhibitors of the invention. In brief summary, the expression of natural or synthetic nucleic acids encoding viral inhibitors, is typically achieved by operably linking a nucleic acid of interest to a promoter (which is either constitutive or inducible), and incorporating the construct into an expression vector. The vectors are suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical cloning vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. See, e. g , Sambrook and Ausbel (both supra) .
To obtain high levels of expression of a cloned nucleic acid it is common to construct expression plasmids which contain a strong promoter to direct transcription, a ribosome binding site for translational initiation (where a polypeptide is to be synthesized), and a transcription/translation terminator. For example, as described herein, the inhibitors of the present invention are optionally expressed in bacterial cells such as E. coli . Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway as described by Yanofsky, C., 1984, ~. Bacteriol., 158:1018-1024 and the leftward promoter of phage lambda 35 (PL) as described by Herskowitz and Hagen, 1980, A2ln. Rev Genet., 14:399-445. The inclusion of selection markers in DNA vectors transformed in bacteria such as E. coli is W O 97/07808 PCTrUS96/12991 also use~ul. Examples of such markers include yenes speci~ying resistance to ampicillin, tetracycline, or chloramphenicol. See, Sambrook, Ausbel, and Berger ~or details concerning selection markers, e.g., for use in E.
coli. Expression systems ~or expressing nucleic acids and polypeptides are available using E. coli, Bacillus sp. (Palva, I. et al., 1983, Gene 22:229-235; Mosbach, K.
et al., Nature, 302:543-545) and Salmonella. E. coli systems are the most common, and best de~ined expression systems and are, therefore, pre~erred.
Methods of transfecting and expressing genes in eukaryotic cells are also known in the art. For example, synthesis o~ heterologous nucleic acids in yeast is well known and described. See, e.g., Sherman et al. (1982) Methods in Yeast Genetics, Cold Spring Harbor Laboratory. Examples o~ promoters for use in yeast include GAL1,10 (Johnson and Davies (1984) Mol. Cell.
Biol 4:1440-1448) ADH2 (Russell et al. (1983) ~. Biol.
Chem. 258:2674-2682), PH05 (EM~O J. (1982) 6:675-680), and MF~l (Herskowitz and Oshima (1982) in The Molecular Biology of the Yeast Saccharomyces (eds. Strathern, Jones, and Broach) Cold Spring Harbor Lab., Cold Spring Harbor, N.Y., pp. 181-209). A multicopy plasmid with selective markers such as Leu-2, URA-3, Trp-1, and His-3 is also commonly used. A number of yeast expression plasmids such as YEp6, YEpl3, YEp4 can be used as expression vectors. These plasmids have been ~ully described in the literature (Botstein et al. ( 1979) Gene 8:17-24; Broach, et al. (1979) Gene, 8:121-133).
Two procedures are commonly used in trans~orming yeast cells. In one case, yeast cells are first converted into protoplasts using zymolyase, lyticase or glusulase, followed by addition of DNA and polyethylene glycol (PEG). The PEG-treated protoplasts are then regenerated in a 3~ agar medium under selective conditions. Details of this procedure are given in Beggs (1978) Nature (London) 275:104-109, and Hinnen et al.

W O 97/07808 PCT~US96/12991 (1978) Proc. Natl. Acad. Sci. USA 75:1929-1933. The second procedure does not involve removal of the cell wall. Instead the cells are treated, e.g., with lithium chloride or acetate and PEG and put on selective plates (Ito, et al. (1983) J. Bact. 153:163-168).
The inhibitors are isolated ~rom yeast by lysing the cells and applying standard nucleic acid (or protein, where appropriate) isolation techniques to the lysates. The nucleic acids o~ this invention are purified to substantial purity by standard techniques well known in the art, including selective precipitation with such substances as ammonium sulfate, isopropyl alcohol, ethyl alcohol, column chromatography, immunopurification methods, and others. See, ~or instance, Sambrook supra, Ausbel supra, and Scopes (1982) Protein Purification: Principles and Practice Springer-Verlag New York.

Transducing cells with nucleic acids can involve, for example, incubating the cells with viral vectors (e.g., retroviral or adeno-associated viral vectors) containing nucleic acids which encode inhibitors o~ interest with cells within the host range of the vector. See, e.g., Methods in Enzymology, vol. 185, Academic Press, Inc., San Diego, CA (D.V. Goeddel, ed.) (199o) or M. Krieger, Gene Transfer and Expression -- A
Laboratory Manual, Stockton Press, New York, NY, (1990) and the references cited therein. The culture of cells used in conjunction with the present invention, including cell lines and cultured cells ~rom tissue or blood samples is well known in the art. Freshney ( Culture of ~nim~7 Cells, a Manual of Basic Techni~ue, third edition Wiley-Liss, New York (1994)) and the re~erences cited therein provides a general guide to the culture o~ cells.

Illustrative o~ cell cultures use~ul for the production of viral inhibitors include cells o~ insect or m~mm~l ian origin. M~mm~l ian cell systems o~ten will be in the form of monolayers of cells, although m~mm~l ian W O 97/07808 PCT~US96/12991 cell suspensions are also used. Illustrative examples of mammalian cell lines include monocytes, lymphocytes, macrophage, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, W138, BHK, Cos-7 or MDCK cell lines (see, e.g., Freshney, supra) .
As indicated above, the inhibitor, e.g., in the ~orm o~ a plasmid which is used to transform a cell, pre~erably contains nucleic acid sequences to initiate transcription and sequences to control the translation o~
any polypeptide which is also encoded by the vector.
These sequences are re~erred to generally as expression control sequences. When the host cell is o~ insect or mammalian origin, illustrative expression control sequences are obtained ~rom Pol III t-RNA promoters (See, Wong-Staal et al . PCT/US94/05700) the SV-40 promoter (Science (1983) 222:524-527), the HIV LTR promoters, the CMV I.E. Promoter (Proc. Natl Acad. Sci (1984) 81:659-663) or the metallothionein promoter (Nature (1982) 296:39-42). The cloning vector containing the expression control sequences is cleaved using restriction enzymes and adjusted in size as necessary or desirable and ligated with DNA coding for inhibitor by means well known in the art.
As with yeast, when higher animal host cells are employed, polyadenlyation or transcription terminator sequences ~rom known m~mm~l ian genes are ~ypically incorporated into the vector. An example o~ a terminator sequence is the polyadenlyation sequence ~rom the bovine growth hormone gene. Sequences ~or accurate splicing o~
the transcript may also be included. An example o~ a splicing sequence is the VPl intron ~rom SV40 (Sprague et al. (1983) ~. Virol. 45: 773-781).
Additionally, gene sequences to control replication in a particular host cell are incorporated into the vector, such as those ~ound in bovine papilloma virus type-vectors. See, Saveria-Campo (1985), "Bovine Papilloma virus DNA a Eukaryotic Cloning Vector" in DNA

Cloning Vol. II a Practical Approach Glover (ed) IRL
Press, Arlington, Virginia pp. 213-238.
Host cells are competent or rendered competent for transformation by various means. There are several well-known methods of introducing DNA into animal cells.
These include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextran, receptor-mediated endocytosis, electroporation and micro-injection of the DNA directly into the cells.
Transformed cells are cultured by means well known in the art. See, Freshny (supra), and Kuchler et al. (1977) Biochemical Methods in Cell Culture and Virology, Kuchler, R.J., Dowden, Hutchlnson and Ross, Inc. The expressed nucleic acids (and polypeptides, where appropriate) are isolated from cells grown as suspensions or as monolayers. The latter are recovered by well known mechanical, chemical or enzymatic means.
See, Scopes, supra.

Preferred Promoters The inhibitors of the invention are most preferably cloned into gene therapy vectors derived from AAV or HIV for transduction of cells in vi tro. In these vectors, the inhibitors are placed into expression cassettes which direct expression of the active inhibitors (SL II decoys, ribozymes, anti-sense nucleic acid, TAR decoy, transdominant gene and the like).
Ideally, expression of the construct should be sufficiently high to inhibit the growth, infection or replication of the virus against which protection is sought. Accordingly, although the selectlon of a partlcular promoter is not a critical aspect of the invention, strong promoters are particularly preferred promoters for directing expression of the inhibltors in the cell. Preferred promoters include Pol III promoters such as the t-RNA promoters ( e . g ., the tRNAVal promoter;
see, Wong-Staal e~ al. PCT/US94/05700), the HIV-2~ LTR
promoter (See, Genbank accession No. U22047 ~or the complete sequence o~ the HIV-2~ virus) and strong basal promoters known to persons o~ skill, including cellular promoters, such as those which direct expression o~ the cytoskeletal machinery, such as the ~-actin promoter and the tubulin promoter.
In addition to the constitutive promoters mentioned above, strong inducible promoters are also pre~erred. In particular, promoters which are expressed upon entry or replication o~ the virus in the cell are particularly pre~erred. For example, HIV LTR promoters are pre~erred promoters when the virus against which protection is sought is an HIV virus.

Measurinq Viral Inhibition The level o~ virus in a cell culture, cell or whole organism is measured by means known in the art.
Typically, the level o~ virus is measured in a western blot or other immunoassay such as an ELISA, or by per~orming quantitative PCR. In immunoassay ~ormats, the level o~ virus is measured by monitoring the amount o~ a viral protein (or viral capsid) by auanti~ying binding o~
the protein to an immunogenic reagent such as an antibody. In quantitative PCR, the level o~ a viral nucleic acid is measured by monitoring PCR ampli~ication products, and comparing the amount o~ ampli~ied nucleic acid obtained, as compared to a ampli~ication products obtained ~rom ampli~ication per~ormed on a known re~erence nucleic acid.

Making An tibodi es Methods o~ producing polyclonal and monoclonal antibodies are known to those o~ skill in the art, and many anti-viral antibodies are commercially available.
See, e.g., Coligan (l991) Current Protocols in Tmm7~nology W O 97/07808 PCT~US96/12991 Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies:
A Laboratory Manual Cold Spring Harbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, CA, and references cited therein; Goding (1986) Monoclonal Antibodies:
Principles and Practice (2d ed.) Academic Press, New York, NY; and Kohler and Milstein (1975) Nature 256:
495-497. Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors. See, Huse et al.
~1989) Science 246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546. Specific monoclonal and polyclonal antibodies and antisera will usually bind with a KD of at least about .1 mM, more usually at least about 1 ~M, preferably at least about .1 ~M or better, and most typically and preferably, .01 ~M or better.
Frequently, the polypeptides and their corresponding antibodies will be labeled by joining, either covalently or non covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241.
In one preferred class of embodiments, the viral proteins detected when quantifying viral inhibition in the present invention are used for the detection of the virus (such as HIV) in human (or animal, e.g , where the animal is a macaque and the virus is HIV-2 or SIV) patients. For instance, HIV polypeptides are used routinely in western blots for the detection of antibodies to HIV in a patient~s blood, and the reciprocal experiment (for detecting HIV polypeptides in a patient's blood) ls suitable for measuring HIV viral load in a patient's blood. Such tests are well known, and are presently a standard method by which HIV-1 and HIV-2 infections are detected in patient populations. A
variety of immunoassay ~ormats are known and available.
A particular protein can be quantified by a variety of immunoassay methods. For a review of immunoloyical and immunoassay procedures in general, see Stites and Terr (eds.) 1991 Basic and Clinical Immunology (7th ed.). Moreover, the immunoassays of the present invention can be performed in any of several configurations, e.g., those reviewed in Maggio (ed.) (1980) Enzyme Immunoassay CRC Press, Boca Raton, Florida;
Tijan (1985) "Practice and Theory o~ Enzyme Immunoassays,ll Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers B.V., Amsterdam; Harlow and Lane, supra; Chan (ed.) (1987) Immunoassay: A Practical Guide Academic Press, Orlando, FL; Price and Newman (eds.) (1991) Principles and Practice o~ Immunoassays Stockton Press, NY; and Ngo (ed.) (1988) Non isotopic Immunoassays Plenum Press, NY.
Immunoassays often utilize a labeling agent to specifically bind to and label the binding complex formed by the antibody and peptide or capsid. Alternatively, the labeling agent may itself be one o~ the antibodies.
In some embodiments, the labeling agent is optionally a third moiety, such as another antibody, that specifically binds to the capture agent/ polypeptide complex, or to a modified capture group (e.g., biotin) which is covalently linked to the antibody.
For example, where the capture agent is a mouse antibody, the label agent may be a goat anti-mouse IgG, i. e., an antibody specific to the constant region o~ the mouse antibodies.
Other proteins capable of specifically binding immunoglobulin constant regions, such as streptococcal protein A or protein G are also useful as labeling CA 02229749 l998-02-l7 agents. These proteins are normal constituents o~ the cell walls o~ streptococcal bacteria. They exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions ~rom a variety of species. See, 5 generally Kronval, et al., (1973) J Immunol., 111:1401-1406, and Akerstrom, et al., ( 1985) J. Tmm77n 135:2589-2542.
Throughout the assays, incubation and/or washing steps may be required a~ter each combination of reagents. Incubation steps can vary ~rom about 5 seconds to several hours, pre~erably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay ~ormat, analyte, volume o~ solution, concentration o~ capture agent and analyte, and the like.
15 Usually, the assays are carried out at ambient temperature, although they can be conducted over a range of: temperatures, such as 5~ C to 45~C.

Sample Collection and Processing An HIV transcript, antibody or polypeptide is pre~erably quanti~ied in a biological sample, such as a cell, or a tissue sample derived ~rom a patient. In a preferred embodiment, antisera to HIV polypeptides or antibodies against HIV polypeptide are quanti~ied in 25 serum ( See, supra) In another pre~erred embodiment, HIV
nucleic acids are detected in an in~ected patient using which monitor the level o~ viral load by hybridization to viral nucleic acids, or ampli~ied products of viral nucleic acids. For instance, in one embodiment, HIV
nucleic acids in a biological sample are ampli~ied by an in vltro ampli~ication technique (e.g , PCR or LCR) and detected using labeled complementary nucleic acids.
Although the sample is typically taken ~rom a human patient, the assays can be used to detect viral 3 5 polypeptides in cells ~rom eukaryotes in general, in particular in primates such as humans, chimpanzees, gorillas, macaques, and baboons, and rodents such as W O 97/07808 PCT~US96/12991 mice, rats, and guinea pigs. The cells are typically part of a whole organism, or in cell culture.
The sample is pretreated as necessary by dilution in an appropriate buffer solution, or concentrated, if desired. Many standard aqueous buffer solutions employing one of a variety of bu~fers, such as phosphate, Tris, or the like, at physiological pH are appropriate. Cell sorting techniques such as FACS are optionally used to isolate particular cells such as CD4 cells in which the virus needs to be quantitated.

Quanti~ication of Polypeptides, nucleic acids and Antibodies HIV antibodies, polypeptides and nucleic acids of the invention are detected and quantified by any o~ a number of means well known to those of skill in the art.
These include analytic biochemical methods such as spectropho~ometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdif~usion chromatography, and the like, and various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescentassays, and the like. The detection of nucleic acids proceeds by well known methods such as Southern analysis, northern analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting, and af~inity chromatography.

Reduction o~ Non Speci~ic Binding One of skill will appreciate that it is often desirable to reduce non specific binding in immunoassays or nucleic acid assays, and during analyte purification.
Where the assay involves a viral antibody, or other capture agent immobilized on a solid substrate, it is desirable to minlmize the amount of non specific binding to the substrate. Means of reducing such non specific ~ binding are well known to those of skill in the art.
Typically, this involves coating the substrate with a proteinaceous composition. In particular, protein compositions such as bovine serum albumin (BSA), nonfat powdered milk, and gelatin are widely used.

Other Assay Formats 10Western blot analysis can also be used to detect and quantify the presence o~ a polypeptide or antibody (including peptide, transcript, or enzymatic digestion product) in the sample. ~he technique generally comprises separating sample products by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose ~ilter, a nylon filter, or derivatized nylon filter), and incubating the sample with labeling antibodies that specifically bind to the analyte protein (antibody or HIV-2 polypeptide). The labeling antibodies speci~ically bind to analyte on the solid support. These antibodies are directly labeled, or alternatively are subsequently detected using labeling agents such as antibodies (e.g., labeled sheep anti-mouse antibodies where the antibody to an analyte is a murine antibody) that specifically bind to the labeling antibody.
Other assay formats include liposome immunoassays (LIAs), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see, Monroe et al., (1986) Amer. Clin. Prod. Rev. 5:34-41).

W O 97/07808 PCT~US96/12991 Label s Labeling agents include e.g., monoclonal antibodies, a polyclonal antibodies, proteins, or other polymers such as affinity matrices, carbohydrates or lipids. Detection proceeds by any known method, such as immunoblotting, western analysis, gel-mobility shi~t assays, fluorescent in situ hybridization analysis (FISH), tracking of radioactive or bioluminescent markers, nuclear magnetic resonance, electron paramagnetic resonance, stopped-flow spectroscopy, column chromatography, capillary electrophoresis, Southern blotting, northern blotting, southwestern blotting, northwestern blotting, or other methods which track a molecule based upon size, charge or affinity. The particular label or detectable group used and the particular assay are not critical aspects of the invention The detectable moiety can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field o~ gels, columns, solid substrates and ~mmnnoassays and, in general, any label useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g. Dynabeads~), fluorescent dyes (e. g ., ~luorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3H, 12sI, 35S, 14C, or 32p), enzymes ( e . g ., LacZ, CAT, horse radish peroxidase, alkaline phosphatase and others, commonly used as detectable enzymes, either as marker gene products or in an ELISA), nucleic acid intercalators (e. g., ethidium bromide) and colorimetric labels such as colloidal gold or colored glass or plastic (e. g.
polystyrene, polypropylene, latex, etc. ) beads.
The label is coupled directly or indirectly to the desired component of the assay according to methods CA 02229749 l998-02-l7 W O 97/07808 PCTrUS96/12991 well known in the art. As indicated above, a wide variety of labels are used, with the choice o~ label ~ dependlng on the sensitivity required, ease of conjugation o~ the compound, stability requirements, available instrumentation, and disposal provisions.
Non radioactive labels are o~ten attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to a polymer. The ligand then binds to an anti-ligand ( e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a ~luorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, ~or example, biotin, thyroxine, and cortisol, it can be used in conjunction with labeled, anti-ligands.
Alternatively, any haptenic or antigenic compound can be used in combination with an antibody.
Labels can also be conjugated directly to signal generating compounds, e . g., by conjugation with an enzyme or ~luorophore. Enzymes o~ interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include ~luorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelli~erone, etc.
Chemiluminescent compounds include luci~erin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review of various labelling or signal producing systems which may be used, see, U.S. Patent No. 4,391,904, which is incorporated herein by re~erence.
Means o~ detecting labels are well known to those o~ skill in the art. Thus, ~or example, where the label is a radioactive label, means ~or detection include a scintillation counter or photographic ~ilm as in autoradiography. Where the label is a ~luorescent label, it may be detected by exciting the ~luorochrome witn the appropriate wavelength of light and detecting the resulting fluorescence, e.g., by microscopy, visual inspection, via photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomuitipliers and the like. Slmilarly, enzymatic labels may be detec~ed by providing appropriate substrates for the enzyme and detecting the resulting reaction product. Finally, simple colorimetric labels are often detected si~ply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold o~ten appears pink, while various conjugated beads appear the color o~ the bead.
Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence o~ antibodies. In this case, antigen-coated (e.g., HIV polypeptide-coated) particles are agglutinated by samples comprising the target antibodies. In this ~ormat, none o~ the components need be labeled and the presence o~ the target antibody is detected by simple visual inspection.

Non Therapeutic Uses o~ the Invention The nucleic acids of the invention are useful as molecular probes, in addition to their utility as therapeutic agents as described herein. A wide variety of formats and labels are available and appropriate for nucleic acid hybridization, including those reviewed in Tijssen (1993) Laboratory Techni~ues in biochemistry and molecular biology--hybridization with nucleic acid probes parts I and II, Elsevier, New York and Choo (ed) (1994) Methods In Molecular Biology Volume 33- In Situ Hybridization Protocols Humana Press Inc., New Jersey (see also, other books in the Methods in Molecular Biology series); see especially, Chapter 21 of Choo (id) "Detection of Virus Nucleic Acids by Radioactive and Nonisotopic in Situ Hybridization~ and the methods , described infra for the detection of nucleic acids in general.
For instance, gel-mobility shift analysis i9 routinely used to detect nucleic acid-protein interactions in biological samples. Accordingly, in one class of embodiments, the inhibitors of the invention which comprise the SL II sequence are used to detect the presence of Rev in a biological sample. In this assay, the inhibitor is labeled, e.g., by radio-labeling the SL
II nucleic acid, and binding of Rev to the inhibitor is monitored in a standard gel-mobility shift assay.
Detection of Rev binding is an indication that the sample contains a virus such as HIV which expresses Rev. Thus, the inhibitors of the invention are, in addition to their therapeutic utility, useful as diagnostic reagents for the diagnosis of HIV infection. Wong-Staal et al. (1991) Viral And Cellular Factors that Bind to the Rev Response Element in Genetic Structure and Requlation of HIV
~Haseltine and Wong-Staal eds.; part of the Harvard AIDS
Institute Series on Gene Regulation of Human Retroviruses, Volume 1), pages 311-322 and the re~erences cited therein describe gel mobility-shift assays for the detection o~ Rev in biological samples, including human blood.
Other methods for the detection of HIV nucleic acids in biological samples using nucleic acids of the invention include PCR, Southern blots, northern blots, in situ hybridization (including Fluorescent in situ hybridization (FISH), reverse chromosome painting, FISH
on DAPI stained chromosomes, generation o~ Alphoid DNA
probes ~or FISH using PCR, PRINS labeling o~ DNA, free chromatin mapping and a variety of other techniques described in Choo (supra)). A variety of automated soild-phase detection techniques are also appropriate.
For instance, large scale polymer arrays are used for the detection of nucleic acids. See, Tijssen ( supra), Fodor et al. (1991) Science, 251: 767- 777 and Sheldon et al.

W O 97/07808 PCT~US96/12991 (1993) Clinical Chemistry 39 (4): 718-719. The inhibitors of the invention can be adapted to use in the above assays, ~or example by monitoring the hybridization o~
the inhibitor to a viral transcript as an indicator that the viral transcript is present in a sample.
Furthermore, the inhibitors o~ the invention inhibit HIV in~ection and replication in cells which comprise the inhibitors. There~ore, one use ~or the inhibitors o~ the invention is ~or the diagnosis o~ viral in~ection in cells in vi tro or ex vivo. In this diagnostic method, cells suspected o~ being in~ected with a particular virus are separated into two populations.
The ~irst population is trans~ected with a viral inhibitor o~ the invention which inhlbits the suspected virus (e.g., in one embodiment, the suspected virus is HIV, and the inhibitor comprises an SL II sequence and an HIV ribozyme), and the second population is treated identically, except that it is not transfected (i.e., the second population is a control). I~ the ~irst cell population shows enhanced viability compared to the second population, it is an indicator that the ce~ll is in~ected with the particular virus.
The compositions o~ the invention ~urther provide a prophylactic utility. The safety ol handling and maintaining cell cultures is enhanced by incorporating the inhibitors o~ the invention into _he cells o~ the cell culture, because the cells are rendered resistant to pathogenic viruses such as HIV. Because the cells are less likely to be in~ected with a pathogenic virus, workers handling the cells are less likely to contract the virus ~rom the cell culture.

Vectors and Trans-com~lementation Trans active genes rendered inactive in a gene therapy vector are "rescued" by trans complementation to provide a packaged vector. This ~orm o~
transcomplementation is provided by vector packaging cell W O 97/07808 PCT~US96/12991 lines, or by co-infection of a packaging cell with a virus or vector which supplies functions missing from a particular gene therapy vector in trans. For instance, cells transduced with HIV proviral sequences which lack the nucleic acid packaging site located in and around the major ~plice donor site and the qag initiator codon adjacent to the 5' LTR produce HIV trans active components, but do not specifically incorporate HIV
nucleic acids into the capsids produced, and therefore produce little or no live virus. If these transduced "packaging" cells are subsequently transduced with a vector nucleic acid which lacks coding sequences for HIV
trans active functions, but includes an HIV packaging signal, the vector nucleic acid is packaged into an infective HIV capsid and envelope. Carrol et al. (1994) Journal of virology 68(9):6047-6051 describe the construction of packaging cell lines for HIV viruses.
Functions of viral replication not supplied by trans-complementation which are necessary for replication of the vector are present in the vector. In HIV, this typically includes, e.g., the TAR sequence, the sequences necessary for HIV packaging, the RRE sequence if the instability elemen~s of the pl7 gene of gag is included, and sequences encoding the polypurine tract. HIV
sequences that contain these functions include a portion of the 5' long terminal repeat (LTR) and sequences downstream of the 5' LTR responsible for efficient packaging, i.e., through the major splice donor site ("MSD"), and the polypurine tract upstream of the 3' ~TR
through the U3R section of the 3' LTR. The packaging site (psi site or ~ site) is partially located adjacent to the 5' LTR, primarily between the MSD site and the gag initiator codon (AUG) in the leader sequence. See, Garzino-Demo et al. (1995) Hum. Gene Ther. 6(2): 177-184.
For a general description of the structural elements of the HIV genome, see, Holmes et al. PCT/EP92/02787.

The TAR sequence is located in the R portion o~
the 5' LTR. It is the sequence to which the tat protein binds. The sequences necessary for packaging are located in the U5 portion o~ the 5' LTR and downstream o~ it into part o~ pl7, as well as the U3R portion o~ the 3' LTR.
The polypurine tract is the sequence upstream ~rom the 3' LTR site where RNAse H cleaves during plus ("+ 1l ) strand DNA synthesis. It mediates plus strand synthesis.
The primate lentiviruses, including HIV-1, HIV-2 and SIV are structurally and ~unctionally similar.Cognate portions o~ any o~ these viruses can be used in the vectors o~ the present invention, or in trans-complementation assays in a manner similar to that described ~or HIV.
HIV virus-based vectors ~or use in qene thera~v Gene therapy provides a method ~or combating chronic in~ectious diseases such as AIDS, caused by HIV
infection, as well as non-in~ectious diseases such as cancer. Yu et al. (1994) Gene T~erapy 1:13-26 and the references therein provides a general guide to gene therapy strategies for HIV in~ection. See also, Sodoski et al. PCT/US91/04335. Wong-Staal et al., PCT/US94/05700 describe HIV-based gene therapy vectors, particularly HIV-1 based vectors.
The primate lentiviruses, including HIV-1, HIV-2 and SIV are structurally and ~unctionally similar.
Cognate portions o~ any o~ these viruses can be used in the vectors o~ the present invention, or in trans-complementation assays as set ~orth herein.
In brie~, when constructing gene therapyvectors ~rom a parental virus, the gene therapy vector is designed so that trans active genes rendered inactive in a gene therapy vector are capable o~
trans-ccmplementation (e.g., by co-cultlvation with the parental viru) in order to render the construct rescuable. This form o~ transcomplementation is used in CA 02229749 l998-02-l7 W 097/07808 PCTrUS96/12991 creating HIV packaging cell lines and in performing co-infection assays and monitoring diagnostic assays and methods described herein. For instance, cells transduced with HIV proviral sequences which lack the nucleic acid packaging site located in and around the major splice donor site and the gag initiator codon adjacent to the 5' LTR produce HIV trans active components, but do not specifically incorporate HIV nucleic acids into the capsids produced, and therefore produce little or no live virus. If these transduced "packaging" cells are subsequently transduced with a vector nucleic acid which lacks coding sequences for HIV trans active functions, but includes an HIV packaging signal, the vector nucleic acid is packaged into an infective HIV capsid and envelope. Carrol et al. (1994) ~ournal of virology 68(9):6047-6051 describe the construction of packaging cell lines for HIV viruses.
Functions of HIV replication not supplied by trans-complementation which are necessary for replication of the vector are present in the vector. This typically includes, e.g., the TAR sequence, the sequences necessary for HIV packaging, the RRE sequence i~ the instability elements of the pl7 gene of gag is typically included, and sequences encoding the polypurine tract. HIV
sequences that contain these functions include a portion of the 5' long terminal repeat (LTR) and sequences downstream of the 5' LTR responsible for e~ficient packaging, i.e., through the major splice donor site ("MSD"), and the polypurine tract upstream of the 3' LTR
through the U3R section of the 3' LTR. The packaging site (psi site) is partially located adjacent to the 5' LTR, primarily between the MSD site and the gag initiator codon (AUG) in the leader sequence. See, Garzino-Demo et al . (1995) HU17l. Gene Ther. 6(2): 177-184. For a general description of the structural elements of the HIV genome, see, Holmes et al. PCT/EP92/02787.

W O 97/07808 PCT~US96/12991 The pl7 gene contains INS (instability) elements that cause rapid degradation o~ the LTR
promoter-mediated transcript in the absence o~ the Rev-RRE interaction. There~ore, i~ the INS sequences are included in the vector, the RRE is also typically included. However, i~ the HIV portion does not contain the INS sequence o~ pl7, then the RRE sequence is optionally omitted. RRE is normally located in the envelope gene o~ HIV and is the sequence to which the rev protein binds.
The TAR sequence is located in the R portion o~
the 5' LTR. It is the sequence to which the tat protein binds. The sequences necessary ~or packaging are located in the U5 portion o~ the 5' LTR and downstream of it into part o~ pl7, as well as the U3R portion of the 3' LTR.
The polypurine tract is the sequence upstream from the 3' LTR site where RNAse H cleaves during plus ("+") strand DNA synthesis. It mediates plus strand synthesis.
Several HIV-2 isolates suitable ~or construction o~ gene therapy vectors have been isolated, including three molecular clones o~ HIV-2 (HIV-2RoD, HIV-25~L-ISY~ and HIV~2ucl), that are reported to in~ect macaques (M. mulatta and M nemestrina) or baboons (Franchini, et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2433-2437;
Barnett, et al (1993) Journal of Virology 67, 1006-14;
Boeri, et al. (1992) Journal of Vlrology 66, 4546-50;
Castro, et al. (l991) Virology 184, 219-26; Franchini, et al. (1990) Journal of Virology 64, 4462-7; Putkonen, et al. (1990) Aids 4, 783-9; Putkonen, et al. (1991) Nature 352, 436-8). HIV-2~ (see supra) also in~ects macaques and human cells, and gene therapy vectors using the HIV-2~ LTR regions are one class o~ pre~erred gene therapy vectors. Another class o~ pre~erred gene therapy vectors includes HIV-1 LTR sequences.

Murine Retroviral Vectors Murine retrovlral vectors are known in the art.
The majority of the approved gene transfer trials in the United States rely on replication-defective retroviral vectors derived from murine retroviruses such as murine moloney retrovirus (referred to alternately as MoLv MoMuLv or MuLv in the art). See Miller et al. (1990) Mol.
Cell. Biol. 10:4239; Kolberg R (1992) J. NIH Res. 4:43, and Cornetta et al. (1991) Hum. Gene Ther. 2:215. The major advantage of murine retroviral vectors for gene therapy are the high efficiency of gene transfer into certain types of replicating cells, the precise integration of the transferred genes into cellular DNA, and the lack of further spread of the sequences after gene transfer.

AAV Vectors Adeno associated viruses (AAVs) require helper viruses such as adenovirus or herpes virus to achieve productive infection. In the absence of helper virus functions, AAV integrates (site-specifically) into a host cell's genome, but the integrated AAV genome has no pathogenic effect. The integration step allows the AAV
genome to remain genetically intact until the host is exposed to the appropriate environmental conditions (e.g., a lytic helper virus), whereupon it re-enters the lytic life-cycle. Samulski (1993) Current Opinion in Genetic and Development 3:74-80 and the references cited therein provides an overview of the AAV life cycle.
AAV-based vectors are used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and in in vivo and ex vivo gene therapy procedures. See, West et al.
(1987) Virology 160:38-47; Carter et al. (1989) U.S.
35 Patent No. 4,797,368; Carter et al. WO 93/24641 (1993);
Kotin (1994) Human Gene Therapy 5 :793-801; Muzyczka CA 02229749 l998-02-l7 (1994) J. Clin. Invst. 94:1351 and Samulski ~supra) i~or an overview o~ AAV vectors.
Recombinant AAV vectors (rAAV vectors) deliver ~oreign nucleic acids to a wide range o~ m~3mm~l ian cells (Hermonat & Muzycka ( 1984) Proc Natl Acad Sci USA
81:6466-6470; Tratschin et al. (1985) Mol Cell Biol 5:3251-3260), integrate into the host chromosome (Mclaughlin et al. (1988) ~J Virol 62: 1963-1973), and show stable expression o~ the transgene in cell and animal models (Flotte et al . (1993) Proc Natl Acad Sci USA 90:10613-10617). Moreover, unlike some retroviral vectors, rAAV vectors are able to in~ect non-dividing cells (Podsako~ et al. (1994) IJ Virol 68:5656-66; Flotte et al. (1994) Az71. J. Respir. Cell Mol. Biol. 11:517-521).
Further advantages o~ rAAV vectors include the lack o~ an intrinsic strong promoter, thus avoiding possible activation o~ downstream cellular sequences, and their naked icosohedral capsid structure, which renders them stable and easy to concentrate by common laboratory techniques. rAAV vectors are used to inhibit, e . g., viral in~ection, by including anti-viral transcription cassettes in the rAAV vector which comprise an inhibitor o~ the invention.

Viral Inhibitors and Gene Thera~y Common gene therapy vectors include those derived from murine retroviruses (including MuLv), avian rous sarcoma virus (RSV), Hepatocyte viruses, HIV-1, HIV-2 and AAV-based vectors. HIV based vectors and AAV based vectors are pre~erred, because they do not require actively dividing cells ~or in~ection (unlike many murine retroviruses). HIV vectors are most pre~erred ~or treating HIV in~ections, because they typically only in~ect CD4~ cells in vivo, i . e., those cells which are 3 5 in~ected by HIV viruses.
The present invention provides several ~eatures that allow one o~ skill to generate power~ul retroviral CA 02229749 l998-02-l7 W O 97/07808 PCTrUS96/12991 gene therapy vectors against specific cellular targets, in vitro and in vivo, e.g., against CD4~ cells. CD4+
cells are infected by HIV viruses (including HIV-1 and HIV-2). HIV viruses also infect a few other cell-types in vitro which exhibit little or no CD4 expression, such as peripheral blood dendritic cells, follicular dendritic cells, epidermal Langerhans cells, megakaryocytes, microglia, astrocytes, oligodendroglia, CD8~ cells, retinal cells, renal epithelial cells, cervical cells, rectal mucosa, trophoblastic cells, and cardiac myocytes (see, Rosenburg and Fauci 1, supra); the infection of these cell types by HIV in vivo, however, is rare. Lists of CD4~ and CD4- cell types which are infectable by HIV
have been compiled ( see, Rosenburg and Fauci 1 supra;
Rosenburg and Fauci (1989) Adv Immunol 47:377-431; and Connor and Ho (1992) in AIDS: etiology, diagnosis, treatment, and prevention, third edition Hellman and Rosenburg (eds) Lippincott, Philadelphia).
The present invention provides viral inhibitors which comprise Rev binding nucleic acids such as SL II
nucleic acids These nucleic acids are useful as components o~ gene therapy vectors. Retroviral vectors packaged into HIV envelopes primarily infect CD4~ cells, (i.e., by interaction between the HIV envelope glycoprotein and the CD4 "receptor") including non-dividing CD4~ cells such as macrophage. For instance, nucleic acids which encode viral inhibitors are encapsidated into HIV capsids in gene therapy vectors which include an HIV packaging site ( e.g., the ~ site in HIV-1), and typically also include the HIV LTR sequences.
Thus, in one preferred embodiment, the inhibitors of the present invention are incorporated into HIV-based gene therapy vectors which deliver the inhibitors to CD4~ cells in a form which results in stable integration and expression of the inhibitor into the cell. This is accomplished by incorporating cis active nucleic acids (e.g., promoter sequences, packaging sequences, W O 97/07808 PCT~US96/12991 integration or cellular targeting sequences) into the vector, or by using trans active nucleic acids and polypeptides (capsid and envelope proteins and transcription factors) to replicate and package the gene therapy vector into an viral capsid ( e.g., an ~IV-l or HIV-2 capsid and envelope), or ~oth. See, e.g., Poznansky et al. (1991) ~ournal or Virology 65(1): 532-536 and Garzino Dem et al. (supra) for a description of the ability of the region flanking the 5' HIV LTR.
A preferred class of embodiments utilizes HIV-2KR LTR sequences as a component o~ a gene therapy vector.
The LTR sequences of HIV-2KR are particularly useful, because they have a high level of basal promoter activity in CD4 cells, and have no tat or rev requirement for transactivation.
In one embodiment, the inhibitors of the present invention comprise anti-sense nucleic acids which specifically hybridize to a viral nucleic acid, thereby inhibiting the activity of the nucleic acid. Wong-Staal et al. PCT application PCT/US94/05700 (WO 94/26877) and Chatterjee et al. (Science (1992), 258: 1485-1488, hereinafter Chatterjee et al. 1) describe anti-sense inhibition of HIV-l infectivity in target cells using viral vectors with a constitutive expression cassette expressing anti-TAR RNA. Chatterjee et al. (PCT
application PCT/US91/03440 (1991), hereinafter Chatterjee et al. 2) describe viral vectors, including AAV-based vectors which express antisense TAR sequences.
Chatterjee and Wong (Methods, A co~r~n;on to Methods in Enzymology (1993), 5: 51- 59) ~urther describe viral vectors for the delivery of antisense RNA. Yu et al.
(1994) Gene Therapy 1: 13-26 and the references cited therein provides a general guide to gene therapy strategies useful against ~IV infection.

W O 97/07808 PCT~US96/12991 Ex Vivo Therapy Ex vivo methods for inhibiting viral replication in a cell in an organism involve transducing the cell ex vivo with a vector o~ this invention, and introducing the cell into the organism. Cells are typically selected based upon the host range o~ the virus against which an inhibitor is directed. For instance, where the virus is an HIV virus, the cells selected for transfection are typically CD4~ cells such as CD4' T
cells, or CD4~ macrophage isolated or cultured from a patient. Stem cells (e.g., CD34i cells) are particularly preferred target cells ~or transduction and use in ex vivo gene therapy procedures. See, e.g., Freshney et al., supra and the references cited therein, and the discusion provided herein for a discussion o~ how to isolate and culture cells from patients. Alternatively, the cells can be those stored in a cell bank ( e.g., a blood bank). In one class of preferred embodiments, the gene therapy vector utilizes an inhibitor which includes an SL II nucleic acid, and an anti-viral therapeutic agent ( e g , trans-dominant gene, ribozyme, anti-sense gene, and/or decoy gene) which inhibits the growth or replication of a virus (e.g., and HIV virus such as HIV-1). The gene therapy vector inhibits viral replication in any of those cells already infected with the virus, in addition to conferring a protective effect to cells which are not infected by the virus.
In addition, in preferred embodiments, the vector is replicated and packaged into viral capsids such as HIV capsid/envelopes using the viral replication machinery. Typically, the necessary ~unctions ~or encapsidation o~ the vector are supplied in trans by a parental virus which recognizes and packages nucleic acids which contain appropriate packaging sequences.
Thus, a patient infected with a virus such as HIV-1 can be treated for the in~ection by transducing a population of their cells with a vector of the invention W O 97/07808 PCT~US96/12991 and introducing the transduced cells back into the patient as described herein. Thus, the present invention provides a method of protecting cells in vitro, ex vivo or in vivo, and the cells are optionally already infected with the virus against which protection is sought.

In Vi vo Therapy Gene therapy vectors containing nucleic acids of the invention can be administered directly to the organism for transduction of cells in vivo.
A~mi ni stration of gene therapy vectors comprising the viral inhibitors of the invention, and cells transduced with the gene therapy vectors is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells. As described herein, preferred vectors utilize HIV viral particles, but other arrangements are also feasible, such as adeno-associated capsids, naked DNA or RNA forms of the gene therapy vectors, or any of the numerous vectors known in the art ( see, supra) . Gene therapy vectors and cells of the present invention can be used to treat and prevent virally-mediated diseases such as AIDS in patients.
The vectors or cells are administered in any suitable manner, preferably with pharmaceutically acceptable carriers. Suitable methods of administering such vectors or cells in the context of the present invention to a patient are available, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention.

WO 97/07808 PCT~US96/12991 Formulations suitable for oral administration can consist o~ (a) liquid solutions, such as an e~ective amount of the vector dissolved in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount o~ the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet ~orms can include one or more o~ lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, ~illers, binders, diluents, bu~fering agents, moistening agents, preservatives, ~lavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge ~orms can comprise the active ingredient in a ~lavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
The vectors, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. Aerosol ~ormulations can be placed into pressurized accep~able propellants, such as dichlorodi~luoromethane, propane, nitrogen, and the like.
Suitable formulations ~or rectal administration include, ~or example, suppositories, which consist o~ the vector with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or para~in hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist o~ a combination o~ the vector with a base, including, ~or example, liquid triglyercides, polyethylene glycols, and para~fin hydrocarbons.
Eormulations suitable ~or parenteral administration, such as, ~or example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, bu~ers, bacteriostats, and solutes that render the ~ormulation isotonic with the blood o~ the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Intravenous administration is the pre~erred method o~ administration ~or gene therapy vectors and transduced cells o~ the invention. The formulations o~ vector can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and in some embodiments, can be stored in a freeze-dried (lyophilized) condition requiring only the addition o~
the sterile liquid carrier, ~or example, water, ~or injections, immediately prior to use. For many vectors, this mode o~ administration will not be appropriate, because many virions are destroyed by lyophilization.
Other vectors (e.g., vectors utilizing an AAV capsid, or naked nucleic acids) tolerate lyophilization well.
Extemporaneous injection solutions and suspensions can be prepared ~rom sterile powders, granules, and tablets o~ the kind previously described.
Cells transduced by the vector, e.g., a-s described above in the context o~ ex vivo therapy, can also be administered parenterally as described above, except that lyophilization is not generally appropriate, since cells are destroyed by lyophilization.
The dose administered to a patient, in the context o~ the present invention should be su~icient to e~ect a bene~icial therapeutic response in the patient over time, or to inhibit in~ection by a pathogenic strain W O 97/07808 PCTrUS96/12991 of HIV. The dose will be determined by the e~icacy of the particular vector employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size o~ the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, or transduced cell type in a particular patient. In determining the ef~ective amount o~ the vector to be administered in the treatment or prophylaxis of virally-mediated diseases such as AIDS, the physician needs to evaluate circulating plasma levels, vector toxicities, progression o~ the disease, and the production of anti-HIV antibodies. In general, the dose of a naked nucleic acid composition such as a DNA is ~rom about l ~g to 100 ~g ~or a typical 70 kilogram patient, and doses o~ gene therapy vectors which include viral capsids such as AAV or HIV vectors are calculated to yield an equivalent amount o~ inhibitor nucleic acid.
In the practice o~ this invention, compositions can be administered, ~or example, by intravenous in~usion, orally, topically, intraperitoneally, intravesically or intrathecally. The pre~erred method o~
administration will o~ten be oral, rectal or intravenous, but the vectors can be applied in a suitable vehicle ~or the local and topical treatment of virally-mediated conditions. The vectors o~ this invention can supplement treatment of virally-mediated conditions by any known conventional therapy, including cytotoxic agents, nucleotide analogues and biologic response modifiers.
For administration, inhibitors and transduced cells o~ the present invention can be administered at a rate determined by the LD-50 o~ the inhibitor, vector, or transduced cell type, and the side-e~ects o~ the inhibitor, vector or cell type at various concentrations, as applied to the mass and overall health o~ the patient.

CA 02229749 l99X-02-17 W O 97/07808 PCT~US96/12991 ~lm;ni stration can be accomplished via single or divided doses.

EXAMPLES
The ~ollowing examples are provided by way of illustration only and not by way of limitation. Those o~
skill will readily recognize a variety o~ noncritical parameters which could be changed or modi~ied to yield essentially similar results.

Materials and Methods The ~ollowing materials and methods were used in the examples below.
Construction ol~ chimeric stem-loop II ~RE/riboz~nne vectors pMJT and pOY-1 are Moloney murine leukemia virus vectors carrying either the HIV-1 5' leader sequence specific ribozyme (anti-1~5 ribozyme) or the HIV-1 Rev sequence speci~ic ribozyme (anti-Rev ribozyme), respectively, driven by the internal human t-RNA
promoter (Yamada et al., Virology (1994) 205:121-126; Yu et al, Proc Natl. Acad. Sci. USA (1993) 90:6340-6344).
pdMJT is a construct containing the disabled ~orm o~ the anti-U5 ribozyme, with the CGU at position 24-26 replaced by AAA (Ojwang et al., Proc. Natl. Acad. Sci. USA. (1992) 89:10802-10806). The stem-loop II sequences o~ RRE in HIV-1 MN (7824-7889) were ampli~ied by PCR with the primer pair 5' SL2 (5'-agagatct, GCA, CTA, TGG, GCG, CAG, C-3') and 3'rcSL2 (5'-cgggatcc, GCA, CTA, TAC, CAG, ACA, AT-3'). The PCR product was digested with Bam HI/Bgl II
and then ligated with Bam HI-digested pMJT. A~ter trans~ormation with this plasmid into the E. coli strain DH5~, a clone in which the SL II was linked to the ribozyme sequence in the same orientation was obtained by screening. The ribozyme sequences in this plasmid, designated as pSLMJT, were replaced with the disabled ribozyme or anti-Rev ribozyme at the Bam HI/Mlu I to - generate pSLdMJT or pSLOY-1, respectively.

Generation of Stable cell lines Molt-4/8 cells were transfected with the parental vector, pMJT, pOY-1, pdMJT, pSLMJT, pSLOY-l or pSLdMJT by the liposome-mediated method using DOTAP
(Boehringer Mannheim). Trans~ected cells were selected by growth in G418 (GIBCO) supplemented media as described previously. Resistant Molt-4/8 cells were designated as MLNL6, MMJT, MOY-1, MdMJT, MSLMJT, MSLOY-1 and MSLdMJT, respectively.

HIV-l SF2 infection of MOY-1 and MSLOY -1 cells G418 selected MOY-1, MSLOY-1, and parental Molt-4/8 cells were incubated with infectious HIV-1 SF2 at an input M.O.I. of 0.01 for 2hr and washed twice.
These cells were cultured at an initial concentration of 105 cells/ml in RPMI1640 medium supplemented with 10 fetal cal~ serum (FCS). On days 5 and 8 after infection, the infected cells were split 1:5 with medium to adjust to a cell concentration of approximately 2 x 105/ml. The culture supernatants were collected on days 3, 5, 8, and 11 after infection, and the level o~ HIV-1 p24 antigen was determined by the HIV-l antigen capture ELISA test (Coulter).

Cocultivation of the stable cell lines with HXB2 infected Jurkat cells Jurkat cells chronically infected with HIV-1 HXB2 were washed twice with RPMI 1640. One hundred or one thousand of these cells were suspended in 1 ml oE
RPMI1640 supplemented with 10~ FCS and containing 105 cells each of the stably transduced cell lines. On day 4 after ini~ection, the cells were split to adjust the cell concentration to approximately 2 x los/ml and the W O 97/07808 PCT~US96/12991 cells were split l: 5 with medium every 3 days therea~ter. The culture supernatants were used ~or measurement o~ p24 antigen by the HIV antigen capture ELISA test (Coulter).
Quan ti ta ti ve Compe ti ti ve (QC) ~T PCR
Total cellular RNA was extracted ~rom ribozyme-transduced cells or parental Molt-4/8 cells by the guanidine thiocyanate-phenol /chloro~orm extraction method (Chomczynski et al ., Anal Biochem (1987) 162:156-159) and subsequently treated with deoxyribonuclease I
(RQI DNase; Promega) as previously described (Yamada et al , Gene Therapy ( 1994) 1:38-45). For QC-RT PCR, in vitro transcribed RNA o~ the anti-U5 ribozyme with a tetraloop substitution (5'-ACA, CAA, CAA, GAA, GGC, AAC, CAG, AGA, AAC, ACA,. CGG, ACU, UCG, GUC, CGU, GGU, AUA, W A, CCU, GGU, A-3') was used as competitor RNA. Total cellular RNA (0 5 mg) and the competitive RNA diluted 10 ~old serially (10 ~g to 10 pg) were added to the RT
reaction mixture (~inal volume, 16 ~1) containing lOmM
Tris-HCl (pH 8 3), 90mM KCl, lmM MnCl2, 200mM each of dATP, dGTP, dCTP, and dTTP, 50p moles o~ Rib 2 and 3 units of Tth DNA polymerase (Promega). A~ter the RT
reaction at 60~ C ~or 20 min, 34 ~l o~ PCR bu~er containing 25 mM Tris-HCl, l lmM EGTA, 200mM KCl, 3 75 mM
MgCl2, 50 pmoles o~ Rib 4 (Yamada et al., Gene Therapy (1994) 1:38-45), and 200mM each o~ dATP, dGTP, dCTP, and dTTP was added to each tube and PCR was carried out (94~
C 30s, 50~ C 30s, 72~ C 30s, 30 cycles). Ten ~l o~ each PCR product was subjected to agarose gel (5~ low melting agarose) electrophoresis The expected sizes o~ the ampli~ied products were 61 bp and 52 bp, respectively, ~or the competitor RNA and the test RNA Gel-images a~ter staining with ethidium bromide were scanned by Twain Scan Duo 600 (Mustek) using Color it v3 0 and analyzed using NIH image v 1 54 by Macintosh computer W O 97/07808 PCT,-'~'S96112991 Quantitative Competitive (QC) PCR
106 cells each of MMJT, MdMJT, MSLMJT, or ~ MSLdMJT were suspended in one ml of a DNase treated HIV-1 HXB2 preparation (lo525TcIDso/ml) in a 1.5 ml tube. The infected cells were incubated for 7 h at 37~ C and washed two times wlth RPMI 1640 medium. Five hundred ~l of lysis buffer containing 50mM Tris, 40mM KCl, lmM
dithiothreitol, 6mM MgCl~, 0.45~ NP40, and 200mg/ml proteinase K was added to each tube and incubated ~or 2h at 50~C. The cell-lysates were heated ~or 10 min in boiling water and used as template DNA ~or QC-PCR. In the QC-PCR, a 5' primer, 32P-end-labeled-SK29 corresponding to nt 501-518 in the LTR) and a 3' primer, SK30 (corresponding to nt 605-589 in the LTR) (Ou et al., Science (1988) 239:295-297) was used.
Competitor DNA was prepared as follows: PCR was carried out with HXB2 DNA as template using a 5' primer, X + 5' LTR, which has 18 random bases (X sequences) flanking the 5' end of the HXB2 LTR 516-534 (5'-gat, agc, ggg, tag, cta, gat, GCT, TAA, GCC, TCA, ATA, AAG, C-3') and a 3' primer, SK 30. The PCR product was reamplified with a 5' primer, SK29 + X, which contains X sequences immediately 3' end of the region corresponding to SK29 (5'-ACT, AGT, GAA, CCC, ACT, GCT, gat, agc, ggg, tag, cta, gat,g-3') and a 3' primer, SK 30. The reamplified product was cloned into pUC 19 at the Sma I site and the resultant plasmid (pUC SK29+X/SK30) was used as competitor DNA. Twenty-five ~l each of the cell-lysate and 5 ~l each of different concentrations (103 to 105 copies in 5 ~l ) of the competitor DNA preparation were added to each 0.5 ml tube containing the reaction mixture (total volume 50 ~l). The composition of the reaction mixture ~or the PCR was 50mM Tris-HCl (pH 8.3), 3mM
MgCl2, 40mM KCl, lmM dithiothreitol, 200mM each of dATP, dGTP, dCTP, and dTTP, and 2.5 pmoles of SK29 (5-7.5 x lOs c.p.m.). Condition of the amplification was 95~C/30s;
50~C/30s; 72~C/30s for 25 cycles. Taq polymerase (1.25 units; Promega) was added to each reaction tube a~ter the ~irst denaturation step (95~C/30s) The expected sizes o~ the amplified products were 105bp and 123bp ~or the test PCR product and the competitor DNA product, respectively. A~ter PCR, 3 ~l each o~ the PCR products were loaded onto an 8~ polyacrylamide gel and autoradiographed Images o~ the gel were scanned by Twain Scan Duo 600 (Mustek) with Color it v3.0 The signal-intensity o~ the competitor and test PCR products was analyzed using NIH image v.1.54 and by Macintosh computer.

Exam~le 1: RRE decoy effect o~ the SL II-ribozyme fusion ~NA
To speci~ically ~x~mine the RRE decoy e~ect o~
an SL II-hairpin ribozyme fusion RNA, HIV-1 SF2 was used as a challenge virus ~or cells expressing anti-U5 and anti-Rev ribozymes fused to SL II (~ig. lA). It was previously reported that the HIV-1 SF2 virus is re~ractory to the anti-Rev (OY-1) ribozyme because o~ a single nucleotide substitution o~ G-~U at the cleavage site (Yamada et al., Virology (1994) 205:121-126), while the U5 target sequence is conserved in this virus.
Expression o~ the anti-Rev ribozyme in the MOY-1 cells and the MSLOY-1 cells was observed by RT-PCR as previously described (Yamada et al., Gene Therapy (1994) 1:38-45) As shown in ~ig. lB, only marginal protection against SF2 in~ection was shown in the MOY-1 cells compared with Molt-4/8, consistent with previous data.
However, expression of p24 antigen o~ ~IV-1 was not detected in the MSLOY-1 cells. Thus, the protection in the MSLOY-1 cells was due to an RRE decoy e~ect o~ the ~usion molecule. In contrast, HIV- 1SF expression was inhibited in both MMJT and MSLMJT cells, expressing either the anti-US ribozyme or the SL II-anti U5 ribozyme ~usion molecule (Fig. lC).

WO 97/07808 PCT~US96/12991 Exam~le 2: ~uantitation of Anti-U5 Ri~ozYme Expression in Stable Cell Lines Expression o~ the ribozyme or disabled ribozyme in MJT, dMJT, MShMJT, and MSLdMJT cells was ~mined by RT-PCR as described herein, using the Rib 4/2 primer pair and oligonucleotide probes that would selectively detect the ~unctional or the disabled ribozyme. Ampli~ied products were specifically detected only when PCR was carried out a~ter reverse transcription. Using a 5' primer corresponding to the SL II sequence, expression o~
the SL II-ribozyme ~usion RNA was detected at 25 weeks a~ter transfection in both cell lines. The expression levels o~ the ribozyme in the stable cell lines were then ~mi ned by QC-RT PCR using the Rib 4 and Rib 2 primer pair. Fig 2 shows the inverted gel images a~ter staining with ethidium bromide. The number o~ competitor RNA
molecules that result in equal signal-intensity o~ the amplified products o~ competitor and test RNA was calculated ~rom regression line by the least-squares method. The ribozyme expression level was thus estimated to be 5.3 x 10' - 6.2 x 10' copies/0.5mg o~ total cellular RNA in the ~our cell lines examined (Fig 2). Since the amount o~ total cellular RNA is generally assessed at lmg RNA/105 cells, it is estimated that each cell was expressing approximately 1000-1200 copies o~ ribozyme containing RNA.
These constitutive levels of ribozyme or ~usion RNA expression had no apparent deleterious e~ect on the Molt 4/8 cells, as all trans~ected cell lines and parental Molt 4/8 cells were indistinguishable with respect to cell-growth rate and viability over a period o~ six months, with passage o~ the cells every 4 days.

W O 97/07808 PCTrUS96/12991 Example 3: Protection aqainst cell-cell transmission o~
HIV-1/NXB2 in the ~usion RNA-expressinq cells The relative antlviral potency of the ribozyme and the SL II/U5 ribozyme vectors was compared in a system utilizing cell-associated virus as the challenge agent. Jurkat cells chronically in~ected with HIV-1/HXB2 were cocultured with stable ribozyme-expressing cell lines at di~erent ratios ~or in~ection (1000:1 and 100:1 unin~ected to in~ected cells). Low levels o~ p24 expression was detected in all cultures early, i.e., ~rom the in~ected ~urkat cells directly. The expression of p24 in the MdMJT and MLNL6 increased sharply at day 25 (Fig. 3A) at 1000:1 in~ection or at day lg at 100:1 in~ection (Fig. 3B). Emergence of virus expression in MMJT and MSLdMJT cells was delayed to day 31 at 1000:1 infection, or day 25 at 100:1 infec~ion. Thus, a single antiviral gene (ribozyme or SL II decoy) had a detectable, inhibitory e~ect on viral replication.
Furthermore, the p24 level was kept at a low level in MSLMJT (ribozyme + SL II) at 1000:1 in~ection during the entire culture period o~ 34 days (Fig. 3A). Even at 100:1 in~ection, the increase in p24 level o~ MSLMJT was delayed ~or an additional 3 days (to day 28) compared to MMJT or MSLdMJT. These results indicated that the combination o~ the SL II and ribozyme was more e~ective than either the ribozyme or SL II decoy alone in the inhibition o~ HIV-l.

ExamPle 4: ComParisOn o~ the ribozyme acti~ity in MMJT
and MSLMJT in a ~irst round infection To ~ mi ne the ribozyme activity o~ the i~usion RNA, the reduction in proviral DNA synthesis was measured in the ~irst round o~ replication a~ter viral challenge.
The RRE decoy e~i~ect i5 not relevant in this early part o~ the replication cycle. The proviral DNA level in the stable cell lines was quanti~ied by competitive PCR 7 hours a~ter challenge with HIV-l/H~32. Proviral DNA was W O 97/07808 PCTrUS96/12991 amplified in the presence of~ di:E~erent concentrations of competitor DNA using 32P-end labeled-SK29/SK30 as the primer pair. The autoradiograph after QC-PCR and the results after analysis of the gel images were shown in 5 Fig. 4A and 4B, respectively. The number of molecules oE
added competitor DNA which results in equal signal intensity of the amplified products ~rom the test and competitor DNA was estimated from the regression line by the least-squares method, and should correspond to the 10 proviral DNA copy number in 2xlOs cells. As expected, no dif~erence in the proviral DNA copy number was observed between MdMJT and MSLdMJT, suggesting a lack of effect of the RRE decoy on preintegration events. The DNA copy number ~or MSLMJT was reduced to 1/7 of that for MSLdMJT, 15 whereas that ~or MMJT was reduced to 1/3. Similar QC-PCR
using a primer pair for ,B-globin DNA confirmed that an equal number o~ cells were used to generate the cell lysate ~or the quantitative analyses. This experiment was repeated with similar results. Consequently, the 20 results demonstrated that the SL II-ribozyme ~usion RNA
indeed ~unctioned as a ribozyme, and the reproducible difEerence observed between MSLMJT and MJT cells suggested that the linkage of the SL II sequence ~urther improved the ribozyme activity. The reduction in DNA
25 level in MMJT cells was 10-20 times less than the result described in a previous paper (Yamada et al., Gene Therapy (1994) 1:38-45). This may be due to the fact that a 20-fold higher M.O.I. (0.2 instead of 0.01) was used Eor inEection in the present study.
All publications and patent applications cited in this speci~ication are herein incorporated by re~erence ~or all purposes as if each individual publication or patent application were speci~ically and 35 individually indicated to be incorporated by reference.

CA 02229749 l998-02-l7 W O 97/07808 PCT~US96/12991 Although the ~oregoing invention has been described in some detail by way o~ illustration and example ~or purposes of clarity o~ understanding, it will be readily apparent to those o~ ordinary skill in the art in light of the teachings o~ this invention that certain changes and modi~ications may be made thereto without departing ~rom the spirit or scope o~ the appended claims.

Claims

1. A Rev binding virus inhibitor which encodes a trans-active ribozyme, wherein said trans-active ribozyme comprises a Rev-binding nucleic acid.

2. The inhibitor of claim 1, wherein said Rev-binding nucleic acid is an SL II nucleic acid.

3. The inhibitor of claim 1, wherein said ribozyme is a hairpin ribozyme.

4. The inhibitor of claim 1, wherein said ribozyme comprises a plurality of rev binding nucleic acids.

5. The inhibitor of claim 1, wherein said ribozyme comprises an SL II nucleic acid at the 3' terminus of the ribozyme and a second SL II nucleicacid at the 5' terminus of the nucleic acid.

6. The inhibitor of claim 1, wherein said ribozyme is selected from the group of ribozymes with the sequences of SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, and SEQ ID NO 7.

7. The inhibitor of claim 1, wherein the inhibitor comprises a recombinant transcription cassette which transcription cassette is encoded by a recombinant gene therapy vector.

8. An RNA encoded by the inhibitor of claim 1.

9. The inhibitor of claim 1, wherein said ribozyme cleaves an HIV-1 nucleic acid in a cell in culture.

10. The inhibitor of claim 1, wherein said inhibitor is a nucleic acid encoded by a gene therapy vector, and wherein said gene therapy vector comprises nucleic acids selected from the group consisting of the HIV packaging site and the AAV ITR.

11. A Rev-binding virus inhibitor comprising an SL II rev binding sequence, which inhibitor does not comprise a full-length RRE sequence, and which inhibitor inhibits viral replication in cells in cell culture.

12. The inhibitor of claim 11, wherein said inhibitor comprises an SL II nucleic acid and a ribozyme.

13. The inhibitor of claim 11, wherein said inhibitor comprises the SL II nucleic acid of SEQ ID NO 1.

14. The inhibitor of claim 11, wherein said inhibitor is an RNA.

15. The inhibitor of claim 11, wherein said inhibitor is a targeted anti-HIV chimeric nucleic acid which encodes a ribozyme, which ribozyme cleaves an HIV nucleic acid, wherein the inhibitor provides greater viral inhibition than a transcription cassette expressing the ribozyme alone.

16. The inhibitor of claim 15, wherein said ribozyme is a trans-ribozyme.

17. The inhibitor of claim 11, wherein said inhibitor, when transfected into a cell culture in vitro and expressed in the cell culture, provides inhibition of the Rev binding virus in the cell culture for more than 15 weeks after the transfection.

18. The inhibitor of claim 11, wherein said inhibitor comprises less than 234 contiguous nucleotides of the sequence comprising the Rev RRE.

19. The inhibitor of claim 11, wherein said inhibitor further comprises a hairpin ribozyme.

20. The inhibitor of claim 11, wherein said inhibitor further comprises an anti-sense nucleic acid which specifically hybridizes to a nucleic acid encoded by the Rev-binding virus.

21. The inhibitor of claim 11, wherein said nucleic acid comprises a plurality of SL II binding sites.

22. A recombinant cell which comprises a nucleic acid encoding a Rev-binding virus inhibitor which comprises a Rev binding site and a ribozyme.

23. The recombinant cell of claim 22, wherein said inhibitor comprises an SL II nucleic acid.

24. The recombinant cell of claim 22, wherein said inhibitor, when expressed in a cell culture in vitro, provides viral inhibition to the cellculture for more than 15 weeks.

25. A cell which stably expresses a Rev-binding virus inhibitor which inhibitor comprises an SL II nucleic acid and a ribozyme.

26. The cell of claim 25, wherein said cell is a CD4+ cell selected from the group consisting of monocytes, lymphocytes and macrophage.

27. The cell of claim 25, wherein said cell is present in a mammal.

28. A method of inhibiting a Rev-binding virus in a cell, comprising introducing into the cell an inhibitor which encodes a nucleic acid comprising a ribozyme, wherein said ribozyme comprises a Rev binding site.

29. The method of claim 28, wherein said cell is present in a cell culture.

30. The method of claim 28, wherein said cell is present in a mammal.

31. The method of claim 28, wherein said method further comprises introducing the cell into a mammal.

32. The method of claim 28, wherein the nucleic acid is introduced into the cell by incorporating the nucleic acid into a gene therapy vector and contacting the cell in vitro with the gene therapy vector.

33. The method of claim 28, wherein the nucleic acid is introduced into the cell by incorporating the nucleic acid into a gene therapy vector and contacting the cell in vivo with the gene therapy vector.

34. The method of claim 28, wherein the nucleic acid is introduced into the cell by incorporating the nucleic acid into a gene therapy vector and contacting the cell with the gene therapy vector, wherein said gene therapy vector comprises nucleic acids selected from the group consisting of the HIV~
packaging site and the AAV packaging site in the ITR.

35. The method of claim 28, wherein the nucleic acid comprises an SL II nucleic acid.